Medical Microbiology: Bacteriology L2

Genomics, Microbial Evolution, and Epidemiology

• Definition of a microbial genome: the total genetic content of an organism
  • In the context of bacteria, it includes the chromosome(s) and plasmid(s)
• Definition of genomics: the study of the structure and function of genes, including the analysis of genome sequences and their applications

Sequencing a Microbial Genome

• Four-step process:
  1. Generate sequencing reads
  2. Read-mapping or de novo assembly
  3. Finishing (most accurate method is to PCR sequence across gap regions or use a different platform)
  4. Genome annotation
• DNA sequencing technologies:
  • Oxford Nanopore: single molecule, real-time sequencing
  • Illumina: genome analyser
• Genome sequencing "explosion" due to the rapid decrease in sequencing cost and increase in speed

Genome Annotation

• Process of locating the position of genes in the genome
• Identifies each open reading frame (ORF) in the genome
• ORF: a reading frame >100 codons that is not interrupted by a stop codon
• CDS: protein-coding DNA sequence encoded by a gene
• BLAST (basic local alignment search tool) computer program:
  • Base-by-base comparison of two or more gene sequences
  • Assigns tentative function of gene or protein structure based on BLAST alignment
• Gene function is often inferred from similarity to other genes, but experimental evidence is rarely used to support annotation

Bioinformatics

• Analysis of genomic data using computers
• Addresses novel questions in microbiology, including:
  • Disease transmission and spread
  • Evolution, including drug resistance
  • Pathogenesis and virulence
  • Microbial ecology

Applications of Genomics

• Foundation for other omic studies (transcriptome, proteome, etc.)
• Rational vaccine design: identifying potential vaccine targets
• Novel drug targets: identifying genes/pathways essential for bacterial growth
• DNA sequence-based diagnostics
• Understand variation in populations: population genetics, phylogenetics, and phylogeography

Bacterial Evolution and Population Genetics

• Identifying mutations within a population
• Assessing genetic variation at a population level
• Determining alleles at single nucleotide loci (SNPs)
• Mapping sequence reads to a reference genome

Phylogenetics and Phylogeography

• Inferring phylogeny from a set of genomic sequences
• Estimating evolutionary relationships among a set of genome sequences
• Phylogeography: combining phylogenetics with geographical mapping to infer how bacteria spread
• Applications in epidemiology: following disease transmission and spread

Epidemiology Revision

• Definition: the science that evaluates the occurrence, determinants, distribution, and control of health and disease in a defined human population
• "Genomic epidemiology" links genomics with epidemiology
• Example: contact tracing

Transport Systems in Gram-Negative and Gram-Positive Bacteria

• Two common transport systems found in both Gram-Negative and Gram-Positive bacteria are the General Secretory Pathway (Sec Translocase Pathway) and the Twin Arginine Transport (TAT) pathway.
• The Sec Translocase Pathway is responsible for the secretion of unfolded proteins, and can be a co-translational or post-translational process.
• Proteins targeted for the Sec system have a signal tag, a 20-30 residue sequence, at the N-termini.
• The TAT system allows the export of folded proteins from the cytoplasm, recognizing a twin arginine tag at the N-termini of proteins.

Transport Across the Inner Membrane

• The Sec Translocase Pathway allows the transport of unfolded proteins across the inner membrane.
• The TAT system allows the export of folded proteins from the cytoplasm, recognizing a twin arginine tag at the N-termini of proteins.

Secretion into the Environment

• The Type 5 Secretion System (T5SS) allows the translocation of proteins across the plasma membrane after Sec translocation.
• T5SS proteins contain four key elements: an N-terminal signal tag, an internal passenger domain, a linker region, and a C-terminal β-barrel/translocator domain.
• Insertion of the translocator domain is mediated by the BAM complex within the outer membrane.
• Multiple classes of T5SS exist, some anchor proteins to the surface, and some allow the release of proteins after cleavage.

T1SS and T5SS Proteins Associated with Virulence

• T1SS: α-haemolysin (HlyA of E. coli) is associated with virulence.
• T5SS: IgA1 protease of Neisseria Gonorrhoeae is associated with virulence.
• T5SS: Ag43 of E. coli is associated with virulence.

Transport Systems Which Translocate Proteins into Hosts

• Some Gram-Negative pathogens have evolved dedicated secretion systems that can translocate proteins across 3 membranes (inner/outer/host).
• These secreted proteins are commonly referred to as 'Effectors'.
• The Type 4 Secretion System (T4SS) enables the translocation of DNA or proteins to other bacteria as well as eukaryotic cells.
• T4SS has two main subfamilies: conjugation machineries (DNA and protein transfer via the VirB/VirD system-T4ASS) and effector protein translocation systems (protein transfer via Dot/Icm system T4BSS).

T4SS Subfamilies

• The VirB/VirD system is evolutionarily related to bacterial conjugation systems.
• The Dot/Icm system is responsible for the translocation of 100s of proteins that block, blunt, and subvert host cells.

Secretion into Other Cells: T6SS

• T6SS appears to share structural features with bacteriophage cell puncturing proteins.
• T6SS is a contact-dependent secretion mechanism that acts as a "poisoned spear" to deliver effectors.
• T6SS targets both bacteria and eukaryotic cells.
• Found in ~25% of Gram-negative bacteria, including human and animal pathogens.

T6SS, Inter-Bacterial Combat

• T6SS can be used as a mechanism to compete with other bacteria (bacterial antagonism).
• Injection (stabbing) of toxins into neighbouring bacterial cells.
• 'Attacker' is protected via cognate immunity proteins.
• Can also be used as a form of bacterial retaliation (tit for tat phenomena).

Transport Systems Unique to Gram-Positive Bacteria (T7SS)

• Unique secretion systems exist in Gram-Positive bacteria, such as the Type 7 Secretion system (T7SS).
• Prototypic example from Mycobacterium tuberculosis (related systems also seen in Staphylococcus aureus).
• T7SS transports proteins across the inner membrane and the mycobacterial cell wall, dependent on a C-terminal signal tag.
• Required for virulence.

Summary

• Multiple secretion systems exist (T1SS, T3SS, T4SS, T5SS, T6SS, and T7SS).
• Systems differ in their ability to transport folded/unfolded proteins, signal sequence locations (N and C termini), and target location (host and environment).

Sec and Tat Secreted Proteins and Virulence

• Sec and Tat secreted proteins can contribute to virulence in bacteria.
• Example: NamA autolysins of Listeria monocytogenes contribute to host colonization by remodeling peptidoglycan.
• Example: Phospholipase C of Pseudomonas aeruginosa (PlcH) requires Tat-based secretion to be functional.

Transport Systems in Gram-Negative Bacteria

• Gram-negative bacteria have two membranes (inner and outer) that require unique mechanisms for protein secretion.
• Secretion systems in Gram-negative bacteria can be divided based on where they deposit their proteins.
• There are multiple transport systems that allow the transport of proteins across the outer membrane from both the cytoplasm and periplasmic space.

Types of Secretion Systems

• Type I secretion (T1SS): translocates proteins across the outer membrane in a one-step process.
• Type II secretion (T2SS): translocates proteins across the outer membrane in a multi-step process.
• Type III secretion (T3SS): translocates effectors directly into the cytoplasm of target eukaryotic cells.
• Type IV secretion (T4SS): enables the translocation of DNA or proteins to other bacteria or eukaryotic cells.
• Autotransporter pathway (T5SS): translocates proteins across the outer membrane using a multi-step process.

Transport Systems that Translocate Proteins into Host Cells

• Type III secretion (T3SS) translocates effectors directly into the cytoplasm of target eukaryotic cells.
• Type IV secretion (T4SS) can translocate effectors into host cells.
• Type VI secretion (T6SS) can also translocate proteins into host cells.

Characteristics of Each Secretion System

• T1SS: C-terminal secretion signal, no folded substrate, 2 membranes, found in E. coli and Pseudomonas.
• T2SS: N-terminal secretion signal, folded substrate, 1 membrane, found in Aceintobacter.
• T3SS: N-terminal secretion signal, no folded substrate, 2-3 membranes, found in EPEC, Shigella, Salmonella.
• T4SS: C-terminal secretion signal, no folded substrate, 2-3 membranes, found in Legionella, Coxiella.
• T5SS: N-terminal secretion signal, no folded substrate, 1 membrane, found in Neisseria, E. coli.
• T6SS: secretion signal unknown, partially folded substrate, 2-3 membranes, found in Burkholderia.

Examples of T1SS and T5SS Proteins Associated with Virulence

• T1SS: α-haemolysin (HlyA of E. coli).
• T5SS: IgA1 protease (Neisseria gonorrhoeae), Ag43 of E. coli.

Type III Secretion System (T3SS)

• Found in Gram-negative pathogens, including Salmonella and Yersinia.
• Translocates effectors directly into the cytoplasm of target eukaryotic cells.
• Shares homologues with flagella biosynthesis and assembly systems.
• Effector proteins contain an N-terminal sequence recognized and bound by specific chaperones.
• Chaperones guide effector proteins to the secretion apparatus.

Type IV Secretion System (T4SS)

• Enables the translocation of DNA or proteins to other bacteria or eukaryotic cells.
• Two main subfamilies: conjugation machineries (DNA and protein transfer) and effector protein translocation systems.
• Examples: Helicobacter pylori Cag system and Bordetella pertussis pertussis toxin secretion.

Bacterial Outer Membrane Vesicles (OMVs)

• OMVs can enter human epithelial cells via multiple mechanisms to interact with NOD1 and be cleared by the host
• OMVs can activate other host pathogen recognition receptors (PRRs) to mediate inflammation and pathogenesis in the host

Pathogen Recognition Molecules (PRMs)

• The TLR family of bacterial pathogen recognition molecules (PRMs) includes NOD1/2 and TLR9
• Bacterial products recognized by PRMs include peptidoglycan (PG) and lipopolysaccharides (LPS)

Biogenesis of OMVs

• OMVs can be produced through mechanisms such as blebbing, bacterial lysis, and membrane remodeling
• The Tol-Pal system maintains bacterial cell membrane integrity during OMV biogenesis
• The Tol-Pal cluster consists of inner membrane proteins (TolA, TolQ, TolR), periplasmic protein (TolB), and an outer membrane peptidoglycan-associated protein (Pal)

Functions of OMVs

• OMVs can mediate inflammation and pathogenesis in the host by activating host innate immune receptors (PRRs)
• OMVs can deliver virulence determinants to and into host cells to promote pathogenesis
• Non-pathogenic functions of OMVs include promoting bacterial survival, acquiring nutrients, degrading antibiotics, and transferring resistance

Gram Positive and Gram Negative Bacteria

• Both Gram positive and Gram negative bacteria produce OMVs with varied cargo
• OMVs produced by Gram positive bacteria contain cargo that can mediate inflammation and pathogenesis in the host
• Examples of Gram positive bacteria that produce OMVs include Bacillus anthracis and Mycobacterium tuberculosis

Environmental Factors and OMV Production

• Environmental factors can increase OMV production by bacteria

Lecture Outcomes

• Bacterial OMVs are a novel secretion system used by all bacteria
• OMVs have different structures and cargo in Gram positive and Gram negative bacteria
• Environmental factors can increase OMV production
• OMVs can mediate inflammation in the host by activating host innate immune receptors (PRRs)

Diagnostic Microbiology

• Diagnostic microbiology involves using a range of tests to identify the disease-causing agent.
• The type and handling of clinical specimens are important considerations, as they can affect the accuracy of test results.

Culture Dependent Methods

• Bacterial culture involves the growth of bacteria on physical media, such as liquid or agar plates.
• Growth can depend on factors such as temperature, oxygen levels, time, and media type.
• Selective/differential media are used to isolate or differentiate bacteria from other organisms within a sample.
• Examples of selective/differential media include EMB agar, which inhibits Gram-positive bacteria and differentiates between lactose fermenters, and Baird-Parker agar, which identifies Staphylococcus aureus.

Biochemical Tests

• Biochemical tests are based on the differential features of closely related organisms or those from similar sites.
• Most biochemical tests are based on enzyme production or activity, such as catalase, oxidase, and urease.
• Other tests involve the utilization of carbohydrates or tolerance to particular conditions, such as 6.5% salt.
• Biochemical tests are often rolled into kits for specific organisms, such as API test strips.

Automated Culture Systems

• Automated culture systems, such as BACTEC or BACT/ALERT, are used for blood, sterile fluids, and platelets.
• These systems grow and scan bottles for bacterial growth, alerting when growth is detected.
• VITEK 2 is an automated system that provides both identification and antibiotic susceptibility testing.

Culture Independent Detection Methods

• Microscopy of specimens can be used for direct detection, including staining and direct and indirect immunofluorescence.
• Immune reactions, such as ELISA, can also be used for detection.
• Molecular methods, including PCR, DNA sequencing, and MALDI, can be used for direct detection.

Microscopic Detection

• Direct examination of smears by staining can be used for diagnosis, with a sensitivity of 36.8% for TB diagnosis.
• Immunofluorescence involves the use of fluorescent antibodies that bind directly to the pathogen, which can be viewed under a microscope.

Molecular Detection Methods

• PCR and real-time PCR are used for nucleic acid amplification, often where organisms are difficult to culture or speed of diagnosis is essential.
• Automated systems, such as GeneXpert for TB diagnosis, are available for molecular detection.

Importance of Diagnosis

• Good diagnosis relies on a combined understanding of the disease and correct interpretation of results.
• Diagnostic tests can be classified into culture-dependent and culture-independent methods.

CRISPR-Cas Systems

• CRISPR-Cas systems consist of three essential components: crRNA, trans-activating crRNA, and Cas9 endonuclease.
• CRISPR-Cas is a class 2, type II system.
• CRISPR-Cas systems have three stages: adaptation, biogenesis, and interference.

Three Stages of CRISPR Immunity

• Adaptation (immunisation): the process of acquiring new spacers from invading viruses or plasmids.
• Biogenesis (crRNA maturation): the process of maturing crRNA from precursor RNA.
• Interference (immunity): the process of cleaving target DNA using Cas9 endonuclease.

The PAM Sequence

• PAM (Proto-spacer Adjacent Motif): a sequence present on invading viral or plasmid DNA, but not part of the CRISPR array.
• PAM sequence is essential for Cas9 to recognise the target sequence and cleave it.
• The canonical PAM sequence is 5'-NGG-3', but different Cas9 proteins recognise different PAMs (2-6 base pairs).

Bacteria and CRISPR-Cas Systems

• CRISPR-Cas systems prove that prokaryotic genomes are environmentally tuned.
• Bacteria can adapt by Lamarckian inheritance, keeping track of and inheriting genomic encounters.
• CRISPR-Cas systems suggest that there are many more unknown processes yet to be uncovered in the ~30% of function unknown genes in each prokaryotic genome.

Application of CRISPR

• CRISPR-Cas systems can be harnessed as a genome engineering tool for precise DNA cleavage.
• Applications of CRISPR include infectious disease, functional genomics, and large-scale genetic screens.
• CRISPR-Cas systems have been used to develop new cancer therapies and may enable the curing of inherited diseases.

History of CRISPR Discovery

• CRISPR was first discovered in the 1980s and 1990s as repeat regions in DNA sequences in different microbes.
• The term "CRISPR" was coined in 2002.
• The 2020 Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer A. Doudna for the development of CRISPR-Cas9 genome editing.

Anti-CRISPR Mechanisms

• Anti-CRISPR mechanisms include mutation leading to mismatches and inefficient cleavage, recombination in polylysogenic genomes, anti-CRISPR proteins (e.g. Acr), and phage carrying CRISPR-Cas elements.
• These mechanisms can inhibit the activity of CRISPR-Cas systems.

Bacterial Encounters

• Humans encounter millions of bacteria daily, but most interactions are transient
• The body is a hostile environment for bacteria, with environmental conditions like skin and intestine, mechanical factors like shedding and peristalsis, and a normal flora that makes up 50% of the body's cells
• The immune system also plays a role in preventing bacterial colonization

Molecular Basis of Bacterial Adhesion

• Bacterial adhesion is the process by which bacteria attach to host cells or receptors
• Adhesins are molecules that mediate adherence, including pili/fimbrial and afimbrial adhesins
• Pili/fimbrial adhesins are complex multigene assemblies with repeating subunits, a tip protein, and a secretion system
• Examples of pili/fimbrial adhesins include type I pili (fim), pap, and afa/Dr
• Afimbrial adhesins are single genes with multiple domains, including trimeric autotransporter adhesins (TAAs)

Adhesin Diversity and Immune Evasion

• Pathogens use alternate colonisation factors, additional domains, and anti-immune mechanisms to evade the immune system
• Adhesins are not always expressed and are tightly regulated to reduce exposure to the immune system
• Phase variation and random expression of adhesins also contribute to immune evasion

Escherichia coli Adherence

• Escherichia coli is a Gram-negative bacterium that causes diarrheal disease
• Different pathotypes of E. coli have different adhesin complements, including F-pili, AAF, and dispersin

Pathotypes and Adhesins

• Enteropathogenic E. coli (EPEC) uses Fim, Bfp, LEE, Paa, and LPF adhesins
• Enterotoxigenic E. coli (ETEC) uses Fim, TCP, and CFAs adhesins
• Enterohaemorrhagic E. coli (EHEC) uses Fim, LEE, Paa, ToxB, Efa-1/LifA, and LPF adhesins
• Other pathotypes of E. coli use different combinations of adhesins

Bacterial Invasion and Dissemination Strategies

• Bacterial invasion involves two main mechanisms: zipper and trigger mechanisms
• The zipper mechanism exploits host cell pathways normally used for adhesion of epithelial cells to the extracellular matrix
• The trigger mechanism resembles cell ruffling induced by growth factors and hormones

Zipper Mechanism - Listeria monocytogenes

• Listeria monocytogenes is a Gram-positive, foodborne pathogen that causes CNS infections and maternofetal infections
• Internalization requires the actin cytoskeleton and bacterial protein internalin A (InlA)
• InlA interacts with E-cadherin, which is involved in tight junction formation, leading to receptor clustering and cytoskeletal changes

Zipper Mechanism - Complications

• Listeria monocytogenes has two invasins: InlA and InlB
• Differences in human and animal receptors lead to differential infectivity and ability of Listeria to bind and invade

Trigger Mechanism - Shigella flexneri

• Shigella flexneri is a Gram-negative, waterborne pathogen that causes bacillary dysentery
• Invasion requires a type 3 secretion system (T3SS)
• T3SS secreted proteins directly assist invasion and target the cytoskeleton and host cell GTPases that regulate actin

Trigger Mechanism - Salmonella

• Salmonella is an enteric pathogen that causes diarrhea
• SPI-1 invasion is encoded on Salmonella pathogenicity island 1 and encodes a type 3 secretion system
• T3SS effectors induce membrane ruffling, facilitating internalization

Intracellular Pathogens

• Intracellular pathogens reside inside host cells to avoid the host immune response and gain access to nutrients
• Advantages of intracellular residence include a privileged environment, inaccessibility to attack by complement and antibodies, and ready access to nutrients
• Disadvantages include a hostile environment if the pathogen is not adapted, immune signaling from infected cells leading to apoptosis, and the need to adapt to intracellular survival

Strategies for Intracellular Survival

• Enter and multiply in non-phagocytic cells using induced endocytosis or macropinocytosis
• Enter and multiply in phagocytic cells using phagocytosis
• Three pathways for intracellular survival: intralysosomal, intravacuolar, and cytosolic

Cytosolic Pathogens - Listeria and Shigella

• Invade cells and escape the vacuole using pore-forming toxins
• Recruit host cell cytoskeletal proteins and induce actin polymerization, forming actin tails that propel bacteria into adjacent cells

Actin Tails - Promoting Cell-Cell Spread

• Actin tails promote bacterial spread within organs and tissues
• Drive bacteria into protrusions that are engulfed by neighboring cells, allowing bacteria to avoid host immunity

Intravacuolar Pathogens

• Establish intracellular niches by constructing membrane-encompassed compartments
• Protect cells from cytoplasmic immune sensing molecules
• Must maintain the integrity of the membrane compartment to survive

Legionella pneumophila - Life Cycle

• Legionella pneumophila is a Gram-negative, aerobic rod that lives in amoebae in the environment and human macrophages during infection
• Two lifecycle phases: replicative and infectious/transmissive
• The infectious phase involves flagellated forms that enter host cells and secrete effectors to prevent lysosome fusion and recruit vesicles from the ER and mitochondria

Legionella pneumophila - Vesicular Manipulation

• Effectors manipulate vesicle trafficking and LCV surface molecules
• Prevents LCV-lysosome fusion and recruits vesicles from the ER and mitochondria to make the LCV resemble the Golgi apparatus

Lysis of Host Cells and Metal Ion Acquisition

• Bacterial pathogens can acquire metal ions through four mechanisms: lysis of host cells, direct acquisition of host proteins, secreted molecules, and direct acquisition of metal ions.

Secreted Molecules (Siderophores and Metallophores)

• Secreted molecules, such as siderophores and metallophores, are released into the extracellular environment to directly compete with host proteins for metal ions.
• Siderophores are low-molecular-weight compounds (500-1500 Daltons) that are primarily synthesized by non-ribosomal peptide synthetases (NRPSs) and are regulated by intracellular Fe abundance.
• Examples of siderophores include hydroxamates and catecholates, which have a high formation constant (Kf) for Fe(III).
• Metallophores, such as nicotianamine derivatives, have broad specificity for divalent cations and are not regulated by cellular Fe abundance.

The Siderophore Arms Race

• The siderophore arms race refers to the competition between bacterial siderophores and host siderocalins (e.g., lipocalin-2) for metal ions.
• Bacteria have evolved to modify their siderophores to evade host siderocalins, such as the synthesis of glycosylated derivatives (e.g., salmochelin).

Direct Acquisition of Metal Ions

• Bacteria can acquire metal ions directly from the host environment using high-affinity metal ion transporters, such as ATP-binding cassette (ABC) transporters.
• These transporters consist of an integral membrane channel and a substrate binding protein, which selectively acquire metal ions and exclude chemically similar metals.

Pneumococcal Surface Adhesin A (PsaA)

• PsaA is a critical virulence determinant in Streptococcus pneumoniae that binds manganese (Mn) with high affinity.
• The PsaA protein is part of the PsaBCA ABC transporter system, which is essential for virulence.
• The crystal structures of PsaA bound to Mn and Zn are largely superimposable, but PsaA achieves selectivity for Mn over Zn through reversible binding.

Therapeutic Targets

• Metal acquisition pathways are crucial for bacterial viability and can be targeted for therapeutic interventions.
• Targeting zinc uptake pathways (e.g., ZnuD) and manganese uptake pathways (e.g., PsaA) has shown modest efficacy in reducing bacterial burden during infection.

Summary

• Metal ions are essential micronutrients for all forms of life, and metal acquisition pathways are crucial for bacterial viability.
• Competition for metal ions at the host-pathogen interface can determine disease outcome, and targeting metal acquisition pathways can be an effective therapeutic strategy.

Evasion of the Innate Immune System by Bacterial Pathogens

• Bacterial pathogens use various mechanisms to evade the innate immune system, including:
  • Expressing a capsule to prevent phagocytosis and complement binding
  • Mimicking the host through molecular mimicry
  • Producing biofilms to avoid immune detection
  • Evasive tactics to survive phagocytosis
• Capsules prevent complement binding and phagocytosis of bacterial pathogens by:
  • Preventing C3b binding on the surface
  • Reducing C3 convertase formation
  • Decreasing C5b production and membrane attack complex formation

Steps of Pathogenesis

• To cause disease, bacterial pathogens must:
  1. Enter the body
  2. Colonize the host
  3. Evade host defenses
  4. Multiply and disseminate
  5. Cause damage to the host
• Immune evasion is a crucial step in pathogenesis

Kinetics of Innate and Adaptive Immune Responses

• Bacterial pathogens need to evade immunity upon entry into the host
• Innate immune responses are immediate, while adaptive immune responses are delayed
• Bacterial pathogens aim to avoid detection and clearance by the immune system

Evasion of Adaptive Immune Responses

• Bacterial pathogens can modify key virulence factors to evade adaptive immunity
• Examples of evasion strategies include:
  • Phase variation in Salmonella to switch between flagella genes
  • Antigenic variation in Neisseria gonorrhoeae to change pili antigens

Phase Variation

• Phase variation refers to changes in the expression of important virulence proteins that occur at high frequency
• It allows bacteria to avoid elimination by the host's immune system
• Examples include:
  • Salmonella switching between flagella genes to avoid immune clearance
  • Neisseria gonorrhoeae altering pili antigens through recombination

Antigenic Variation

• Antigenic variation involves changing surface antigens through gene shuffling events
• This allows pathogens to avoid host antibody responses by providing a new antigenic variant
• Examples include:
  • Neisseria gonorrhoeae using antigenic variation to change pili antigens
  • Other bacterial pathogens using similar mechanisms to evade adaptive immunity

Identifying Virulence Factors

• Two recognized mechanisms for bacteria to invade host cells
• One mechanism involves disruption of some Dot/Icm effectors, associated with phenotypes
• Similar studies: Martinez et al., PLoS Pathog, 2014 (cig2::Tn), and Chao et al, 2016, Nat Rev Micro (TnSeq results)

TnSeq Advantages

• Capacity to test very large numbers of mutants at once, statistically very powerful
• Significant reduction in the number of animals used in animal models

Koch's Postulates

• 1. The microbe must be found in all organisms suffering from the disease and not in healthy organisms
• 2. The microbe must be isolated from the diseased organism and grown as a pure culture
• 3. A pure culture of the microbe, when inoculated into a susceptible and healthy host, must reproduce the disease
• 4. The microbe must be reisolated in pure culture from the experimentally infected host

Limitations of Koch's Postulates

• Not applicable in the molecular era

Molecular Era Postulates

• 1. The phenotype or property under investigation should be associated with pathogenic members of a genus or pathogenic strains of a species
• 2. Specific inactivation of the gene(s) associated with the suspected virulence trait should lead to a measurable loss in pathogenicity or virulence
• 3. Reversion or allelic replacement of the mutated gene should lead to restoration of pathogenicity

Identifying Virulence Genes

• Phenotype: understanding disruption of some Dot/Icm effectors
• Model: Transformation and antibiotic selection
• Method: Array into 96-well tray, results from Carey and Newton, PLoS Pathog, 2011

Screening for Virulence Factors

• Current approach: transformation and antibiotic selection
• Limitations: labor-intensive and time-consuming
• Potential improvements: TnSeq, which can test very large numbers of mutants at once, statistically very powerful

Types of Vaccines

• Live attenuated vaccines: weakened or inactivated pathogens that induce similar immune responses to natural infection
• Killed whole organism vaccines: inactivated pathogens that stimulate an immune response
• Toxoid vaccines: detoxified toxins from pathogenic bacteria that neutralize disease symptoms
• Subunit vaccines: use purified protein, recombinant protein, polysaccharide, or peptide from pathogens
• Virus-like particle vaccines: resemble viruses but lack genetic material
• Outer membrane vesicle vaccines: use Gram-negative bacterial outer membrane antigens
• Conjugate vaccines: combine carrier protein with polysaccharide from pathogens
• Viral vectored vaccines: use viral vector to deliver pathogen genes
• Nucleic acid vaccines: use DNA or RNA to stimulate an immune response

Live Attenuated Vaccines

• Show limited replication in vivo but induce strong immune responses
• Resemble natural infection, delivering antigens with infection-specific PAMPs
• One of the most effective methods for generating T cell-based immunity
• Common methods for attenuation include:
  • Using closely related species causing milder disease
  • Long-term cultivation or passaging
  • Chemically induced multi-site mutagenesis

Toxoid Vaccines

• Neutralize toxins produced by pathogenic bacteria
• Detoxified toxins are highly immunogenic but require multiple boosters over lifetime
• Two licensed toxoid vaccines to date: Tetanus toxin (TT) and Diphtheria toxin (DT)
• Main risks and weaknesses include producing toxin and positive selection

Antibiotics Overview

• Antibiotics are molecules that kill or stop the growth of microorganisms, including bacteria, fungi, or viruses.
• Antibiotics can be derived from nature (natural products), synthesized (synthetic chemistry), or semi-synthetic (modified natural products).

Sources of Antibiotics

• Bacteria/Fungi: Actinomycetes (e.g., Streptomyces) produce antibiotics, primarily tetracycline and methicillin.
• Semi-synthetic antibiotics are made by modifying natural products in a lab.

Classifying Antibiotics

• Antibiotics can be classified as bactericidal (kill bacteria) or bacteriostatic (inhibit growth).
• They can also be classified as broad-spectrum (effective against both Gram-positive and Gram-negative bacteria) or narrow-spectrum (effective against either Gram-positive or Gram-negative bacteria).

Ideal Antibiotic Properties

• Irreversible binding to a bacteria-specific target, resulting in bacterial cell death.
• Ability to penetrate Gram-negative and Gram-positive cell walls.
• Activity at very low concentrations.
• No drug-drug interactions.
• Broad spectrum.
• High oral availability.
• Good tissue penetration.
• Limited ability for resistance to develop.

Antibiotics Targeting the Cell Wall

• Antibiotics can target the bacterial cell wall, specifically peptidoglycan.
• Peptidoglycan is composed of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) linked by oligopeptide chains.

Beta-Lactam Antibiotics

• Beta-lactam antibiotics, such as penicillin, inhibit bacterial cell wall synthesis.
• Penicillin inhibits DD-transpeptidase (Penicillin Binding Protein, PBP).
• Vancomycin binds to D-Ala-D-Ala of the stem peptide, inhibiting peptidoglycan synthesis.

Penicillin vs. Vancomycin

• Penicillin: Derived from fungi (Penicillium chrysogenum), discovered by Alexander Fleming in 1928.
• Vancomycin: Derived from Actinobacteria (Amycolatopsis orientalis), discovered through soil screening in 1954.

Antibiotics Targeting the Ribosome

• Antibiotics can target the bacterial ribosome, specifically the 30S and 50S subunits.

Tetracyclines

• Tetracyclines, such as tetracycline and doxycycline, inhibit protein synthesis by binding to the 30S ribosomal subunit.
• They prevent amino-acyl tRNA binding to the A site.

Linezolid

• Linezolid, an oxazolidinone, inhibits protein synthesis by binding to the 50S ribosomal subunit.
• It blocks the P-site and stops initiation of protein biosynthesis.

Tetracyclines vs. Linezolid: Resistance

• Tetracycline resistance is often due to ribosomal methylation or efflux.
• Linezolid resistance is rare, likely due to its novel mechanism of action.

The Problem of Antibiotic Resistance

• Antibiotic resistance is increasing, and it is a WHO priority.
• Priority 1 pathogens include Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae, which are critical to address.
• Priority 2 pathogens include Enterococcus faecium, Staphylococcus aureus, and Helicobacter pylori, which are considered high priority.

Definition of Antibiotic Resistance

• Antibiotic resistance refers to an organism's ability to grow in the presence of an antibiotic designed to kill or inhibit it.
• Resistance can be caused by various factors, including intrinsic and acquired mechanisms.

Importance of Antibiotic Resistance

• Antibiotic resistance leads to untreatable infections, longer hospital stays, greater costs, and increased mortality.
• Several infections are completely untreatable due to antibiotic resistance.

Genetic Basis of Resistance

• Intrinsic resistance can arise from various mechanisms, including the cell type, lack of target or mutated target, and chromosomal resistance genes.
• Acquired resistance can occur through horizontal gene transfer, involving extrachromosomal DNA, plasmids, transposons, and phage.

Origins of Resistance

• Antibiotic producers must protect themselves from their products, leading to the evolution of resistance genes.
• Many antibiotic producers are resistant to multiple antibiotics, suggesting that these genes are ancient.
• The origins of resistance can be traced back to environmental bacteria, with evidence from genomics and permafrost sequencing.

Resistance Mechanisms

• Efflux pumps actively pump antibiotics out of the cell, and many targets are inside the cytoplasm or inner membrane.
• There are five major families of efflux pumps, with different substrate specificities.
• Inactivation mechanisms involve the destruction of antibiotics, such as β-lactamases, which cleave the β-lactam ring.
• Target modification mechanisms involve the modification of the antibiotic target, such as methicillin resistance in Staphylococcus aureus.

Identification of Resistance

• Antibiotic susceptibility testing is used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antibiotics.
• Methods for testing include disk diffusion, E-test, VITEK, and broth microdilution.
• Genomics can also be used to detect resistance markers.

Why Do We Have a Resistance Problem?

• The introduction of an antibiotic is always followed by the emergence of resistance.
• The antibiotic discovery timeline has slowed, with no new antibiotic classes since the 1990s.

What Can We Do About Resistance?

• Antibiotic stewardship involves using fewer antibiotics, only when necessary, and stopping unnecessary use in agriculture.
• Alternative treatments, such as phage therapy, can be used to combat antibiotic resistance.
• New antibiotics can be discovered through natural product screening, whole cell assays, and target-based screening.

Antibiotic Discovery

• Natural products, such as those from Streptomyces, are a rich source of antibiotics, but it is challenging to identify new structures and access complex gene clusters.
• New methods, such as iChips, induction, and clone and express genes, can be used to overcome these challenges.
• Antibiotic discovery from nature is an excellent source, but it is challenging to identify new structures and access complex gene clusters.

ESKAPE Pathogens

• ESKAPE pathogens are a group of bacteria that are highly resistant to antibiotics and cause serious infections in healthcare settings.
• The acronym ESKAPE stands for:
  • E: Enterococcus faecium
  • S: Staphylococcus aureus
  • K: Klebsiella pneumoniae
  • A: Acinetobacter baumannii
  • P: Pseudomonas aeruginosa
  • E: Enterobacter spp.

Staphylococcus aureus

• S. aureus is a gram-positive, cocci-shaped bacterium that can cause a range of acute and invasive infections.
• Its genome is enriched with mobile genetic elements, which allows for the transmission of virulence factors, including antibiotic resistance.
• Methicillin resistance in S. aureus arises from the mecA gene, which is carried on the mobile genetic element SCCmec.

Methicillin-Resistant S. aureus (MRSA)

• MRSA emerged in 1960, shortly after the introduction of methicillin.
• MRSA remained rare until the 1980s, but its prevalence has increased significantly since then.
• The mecA gene confers resistance to almost all β-lactam antibiotics and is transmitted on mobile genetic elements.

Enterococcus faecium

• E. faecium is a gram-positive, cocci-shaped bacterium that is a commensal organism in the gastrointestinal tract of humans and animals.
• However, it can also cause healthcare-associated infections (HAIs) and has acquired tailored gene repertoires not present in commensal organisms.
• The genome of E. faecium is highly dynamic, with a prominent feature being the constant flux of accessory genes through horizontal gene transfer events.

Vancomycin Resistance

• Vancomycin resistance in E. faecium is carried on mobile genetic elements, which can be transmitted to other bacteria.
• The vanB operon on Tn1549 is mobilized via horizontal gene transfer within the gastrointestinal tract to E. faecium.
• De novo evolution of vanB-mediated resistance has also been observed in patients.

WHO Priority Pathogens

• The World Health Organization (WHO) has classified pathogens into three priority categories for the development of new antibiotic therapeutics:
  • Critical Priority: Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae.
  • High Priority: Enterococcus faecium, Staphylococcus aureus, and Klebsiella pneumoniae.
  • Medium Priority: Streptococcus pneumoniae, Haemophilus influenzae, and Shigella spp.

Acinetobacter baumannii and Pseudomonas aeruginosa

• Two Gram-negative bacterial pathogens that impact human health
• Both are opportunistic pathogens and major causes of nosocomial infections

Acinetobacter baumannii

• Gram-negative, short rod-shaped bacterium
• Motile, but lacks flagella; uses Type IV pili or secreted exopolysaccharide
• Strict aerobe
• Classified in the Gamma-proteobacteria
• Evolved from a low-virulence commensal bacterium to a successful opportunistic pathogen
• Environmental reservoir is unclear, with some studies suggesting it is ubiquitous in the environment, while others argue it is solely present in hospital environments
• Relatively large bacterial genome, averaging 3.5 Mbp, with a core genome of only 1,344 genes
• Pan-genome contains large amounts of non-conserved material, indicating horizontal gene transfer
• Clinically significant opportunistic pathogen, causing pneumonia, UTIs, wound infections, meningitis, and bacteremia, particularly in ICUs and aged-care facilities
• Treatment options are limited, with increasing carbapenem-resistance, and phage-therapy showing some success

Pseudomonas aeruginosa

• Gram-negative, encapsulated, rod-shaped bacterium
• Unipolar motility, using flagella and Type IV pili
• Classified in the Gamma-proteobacteria
• Secretes pigments, including pyocyanin, pyoverdine, pyorubin, and pyomelanin
• Ubiquitous environmental bacterium, present in soil, water, skin flora, and man-made environments
• Large bacterial genome, averaging 6.2 Mbp, with a core genome of only 665 genes
• Horizontal gene transfer accounts for much of the pan-genome
• Facultative anaerobe, able to grow in microaerophilic or anaerobic conditions
• Clinically significant opportunistic pathogen, particularly in immunocompromised individuals, causing respiratory tract, urinary tract, and blood infections
• Frequent colonizer of aseptic medical devices, with high mortality rates associated with pneumonia and bacteremia

Cystic Fibrosis and Pseudomonas aeruginosa

• P. aeruginosa is a leading cause of morbidity and mortality in cystic fibrosis patients
• The bacterium rapidly adapts to the mucus in the lungs of CF patients, leading to chronic infection
• Strains isolated from CF lungs have dramatic genetic, morphological, and physiological changes, rarely seen in non-CF individuals
• Characteristics of CF clinical P. aeruginosa isolates include mucoidy, lack of virulence factor expression, hyper-mutability, and resistance to multiple classes of antibiotics

Antibiotic Resistance

• A. baumannii and P. aeruginosa have low antibiotic susceptibility due to multifactorial resistance mechanisms
• Mechanisms include multidrug efflux pumps, antibiotic-degrading and -inactivating enzymes, low permeability of cellular membranes, and rapid acquisition of new resistance determinants
• Resistance to common first-line antibiotics, including β-lactams, carbapenems, polymyxins, and aminoglycosides, limits treatment options
• Decreasing pipeline of new antibiotics and increasing antibiotic resistance is a major concern

Resistance-Nodulation-Division (RND) Family

• Ubiquitous in bacteria, archaea, and eukaryotes
• Efflux pumps that are highly prevalent in many Gram-negative bacterial species
• Function as proton/substrate antiporters, with 7-8 phylogenetic families
• RND systems involved in drug efflux are AdeABC, AdeFGH, and AdeIJK in A. baumannii, and 13 RND transport systems in P. aeruginosa

MST Review

• The overall score of the class is 29/35 (85%) with a standard deviation of 3.5 (10.1%) and a median score of 31 (88.6%).
• 100% of the class passed, with ~3/4 scoring above 80%.
• Questions that were answered well include diagnostic tests, Koch's postulates, and vaccines.
• Questions that were not answered well include "choose the incorrect answer" type questions, and some questions were left unanswered.
• Tips for short answer questions: read the question, avoid "brain dumping", and provide clear explanations with factual backup.

MST 2

• The MST 2 test will take place on Friday, 3rd May at 10am, lasting 45 minutes and worth 20% of the final mark.
• The test will consist of short and extended answer questions, covering lectures 14-26, with no multiple-choice questions.

Hospital Acquired Infections

• Colonization vs infection: understanding the difference is crucial.
• Study methods used to track hospital acquired infections include surveillance and tracing.
• Limitations of studies on hospital acquired infections include differences in surveillance methods, regional differences in data sources, and limited evidence of clonal relationships between colonizing and infecting strains.

Paper 1

• The study investigated the difference between colonization and infection.
• The study found various results, but limitations included differences in surveillance methods and regional differences in data sources.

Paper 2

• Multidrug-resistant S. epidermidis is a potential problem in hospitals.
• The mechanism of resistance, especially to vancomycin, in S. epidermidis is important to understand.
• Clonal expansion of MDR S. epidermidis in hospitals is a concern.

Scenario

• To determine the origins, risk factors, and potential outbreak of a hospital pathogen, track the spread of the organism within the hospital.
• Methods to track the spread include:
  • Determining if the organism has a hospital or community origin.
  • Identifying factors involved in spreading the organism within the hospital.

Bacterial Toxins

• Bacterial toxins are toxic substances produced and released by bacteria to target other bacterial or host cells.
• Can be classified into two main types: endotoxins and exotoxins.

Endotoxins

• Lipopolysaccharides associated with the cell wall of Gram-negative bacteria.
• Integral part of Gram-negative bacterial cell wall, mainly released when bacteria are lysed.
• Consists of three parts: O antigen, core, and Lipid A.
• Lipid A is responsible for toxicity, and TLR4 is the LPS receptor.
• Pyrogenic, less potent than exotoxins, and can lead to fever, diarrhea, and septic shock.

Mechanism of Endotoxin

• Endotoxin activates macrophages, which produce IL-1 and TNF.
• Leads to the activation of complement and the coagulation cascade.
• Results in hypotension, fever, and inflammation.

Effects of Endotoxin

• Fever caused by the production of IL-1 by macrophages.
• Leukopenia followed by leukocytosis.
• Hypotension, an early marker of Gram-negative bacteremia.
• Septic shock, which can lead to organ failure and death.

Exotoxins

• Produced by Gram-positive and Gram-negative bacteria.
• Highly potent, and avirulent strains have often lost the ability to make toxins.
• Generally released or secreted from the bacterial cell.
• Most have a specific mode of action, and can be classified according to their activity.

Exotoxin Classification

• Classified according to their host cell tropism (e.g., cytotoxin, haemolysin, leukotoxin).
• Classified according to their site of action (e.g., neurotoxin, cardiotoxin, enterotoxin).
• Classified according to the disease or bacteria (e.g., diphtheria toxin, anthrax toxin).
• Classified according to their enzymatic activity (e.g., mono glucosyltransferase, ADP-ribosyltransferase).

A-B Toxins

• Consist of two components: one component (subunit A) is responsible for the activity, and one component (subunit B) is responsible for binding.
• Generally, the "A" subunit is not active until it is released into the cell by subunit "B".
• Can be single-chained or multi-chained.

Examples of A-B Toxins

• Diphtheria toxin: ADP-ribosylation.
• Exotoxin A: ADP-ribosylation.
• Botulinum toxin: Zn2+ protease.
• Tetanus toxin: Zn2+ protease.
• Cholera toxin: ADP-ribosylation.
• Shiga toxin: cleaves 23S rRNA.
• Anthrax toxin LF: Zn2+ protease.
• Anthrax toxin EF: adenylate cyclase.
• CDT binary toxin: ADP-ribosylation.

Diphtheria Toxin

• Produced by Corynebacterium diphtheria.
• Causative agent of diphtheria.
• Encoded by a prophage.
• First identified toxin, discovered in 1888 by Émile Roux and Alexandre Yersin.
• Lethal dose in humans is 100 ng/kg.
• Causes inflammation of the heart muscle and nerves.

ADP-Ribosyltransferases

• ADP-ribosyltransferase is a common activity of exotoxins.
• Transfer of an ADP-ribose moiety from NAD+ to target proteins of intoxicated eukaryotic cells.
• Inhibits the function of target proteins.

Examples of ADP-Ribosyltransferases

• Diphtheria toxin: inhibits protein synthesis.
• Pseudomonas exotoxin A: inhibits protein synthesis.
• Cholera toxin: activates adenylate cyclase.
• TEC heat-labile toxin: activates adenylate cyclase.
• Pertussis toxin: activates adenylate cyclase.
• C.botulinum C2 toxin: inhibits actin polymerization.
• S.aureus EDIN toxin: disassembles Golgi.
• CDT binary toxin: inhibits actin polymerization.

Anthrax Toxin

• Produced by Bacillus anthracis.
• Causative agent of anthrax.
• Encoded by a plasmid.
• Anthrax toxin is a multi-chain A-B exotoxin.
• Made up of three proteins: Protective Antigen (PA), Edema Factor (EF), and Lethal Factor (LF).

Mechanism of Anthrax Toxin

• EF converts cellular ATP to cAMP, leading to an increase in fluid.
• LF inactivates MAPKK, leading to apoptosis and death.

Not all Exotoxins are Proteins

• Recently, several non-protein toxins have been identified.
• Not all exotoxins are proteins.

Clostridial Diseases

• The genus Clostridium is diverse, consisting of Gram-positive, obligate anaerobes that form heat-resistant endospores.
• While not all Clostridium species are pathogenic, several are important human and animal pathogens.
• Pathogenic Clostridia can be classified into three categories: neurotoxic, enterotoxic, and histotoxic.

Neurotoxic Clostridia

• Clostridium tetani causes tetanus.
• Clostridium botulinum causes botulism, a disease that targets the nervous system.
• Botulinum toxin (BoNT) is the most potent biological toxin known, causing flaccid paralysis and potentially death.

Enterotoxic Clostridia

• Clostridiodes difficile (C. diff) is a leading cause of infectious diarrhea in hospitals worldwide.
• C. diff spores are essential for transmission and persistence in hospitals.
• C. diff infections can cause a spectrum of diseases, including self-limiting diarrhea, pseudomembranous colitis, toxic megacolon, sepsis, and death.

Virulence Factors of C. diff

• The major virulence factors of C. diff are two toxins: TcdA (Toxin A) and TcdB (Toxin B).
• Both toxins are members of the large clostridial toxin (LCT) family.
• TcdA and TcdB are encoded in the Pathogenicity Locus (PaLoc).
• TcdA and TcdB are monoglucosyltransferases that glucosylate Rho family GTPases, disrupting the actin cytoskeleton and causing cell death.

Clostridium perfringens

• C. perfringens is an important pathogen of humans and animals, causing diseases such as clostridial myonecrosis (gas gangrene) and food poisoning.
• Historically, C. perfringens was typed according to the toxin profile of infecting strains (A-E).
• C. perfringens produces up to 17 different toxins, including α-toxin, β-toxin, and ε-toxin.
• α-toxin is essential for gas gangrene, and its mechanism of action involves phospholipase C activity, cleaving cell membrane phosphatidylcholine to phosphorylcholine and diacylglycerol.

C. perfringens Food Poisoning

• C. perfringens is a major cause of food poisoning globally, with symptoms including abdominal cramping, diarrhea, and vomiting.
• Ingestion of C. perfringens enterotoxin (CPE) producing strains in under-cooked food can cause food poisoning.
• CPE is a pore-forming toxin of the aerolysin family, binding to claudin receptors and inducing oligomerization and pore formation, leading to tight junction damage and disruption of epithelial permeability barrier.

Chlamydia

• Chlamydia is the most common STI in the world, with over 200,000 cases in the UK in 2017.
• Caused by Chlamydia trachomatis, an exclusive human pathogen.
• Asymptomatic in 80% of individuals, but can lead to blocked fallopian tubes and infertility.
• Transmitted via sexual contact and vertical transmission.
• Recognized as a specific STI in the 1970s, with little to no history before this time.

Chlamydia Disease Symptoms

• In females:
  • Cervicitis, urethritis, pelvic inflammatory disease, perihepatitis, or proctitis.
  • Untreated, increases risk of infertility and ectopic pregnancy.
  • Infants born vaginally to mothers with genital Chlamydia trachomatis may develop conjunctivitis and/or pneumonia.
• In males:
  • Urethritis, epididymitis, prostatitis, proctitis, or reactive arthritis.
• In both sexes:
  • Conjunctivitis, pharyngitis, and lymphogranuloma venereum (enlarged lymph nodes).

Chlamydia Trachomatis Serovars

• 18 serovars that correlate with multiple medical conditions:
  • Serovars A, B, Ba, and C: ocular infection → chronic conjunctivitis, can cause blindness.
  • Serovars D-K: Genital tract infections, neonatal infections.
  • Serovars L1-L3: Lymphogranuloma venereum (LGV).

Chlamydia Trachomatis Life Cycle

• Two developmental forms:
  • Elementary body (EB): infectious form, taken up by host cells.
  • Reticulate body (RB): differentiates from EB, replicative form inside host cells.
• EBs:
  • Spherical, ~0.2 μm diameter.
  • Osmotically stable, less permeable than RBs → environmentally resistant.
  • Preloaded with virulence factors.
  • Internalized following epithelial attachment, enter a vacuole, known as an inclusion.
• RBs:
  • Spherical, ~0.8 μm diameter.
  • Not infectious.
  • Not stable outside of the host cell.
  • Actively replicate inside the host cell.
  • Enriched in proteins involved in nutrient uptake, ATP generation, and translation.

Chlamydia Trachomatis Intracellular Niche

• The inclusion:
  • Migrates to microtubule organizing centre.
  • Regulates fusion and membrane dynamics.
  • Inhibits lysosome fusion, but promotes fusion with nutrient-rich vesicles.
  • Scavenges nutrients, especially lipids.
  • Interacts with other organelles, such as lipid droplets, peroxisomes, and mitochondria.

Chlamydia Trachomatis Modifying the Host Response

• Premature host-cell death limits replication.
• Chlamydia:
  • Activate pro-survival pathways and inhibit apoptotic pathways.
  • Slow progression of the host cell cycle.
  • Dampen DNA damage response.
  • Induce inflammatory response, but block NF-kB activation.
  • Modify host transcriptome and proteome.

Chlamydia Trachomatis Genomics

• First genome finished by Sanger sequencing in 1998.
• Very small genome: 1.04 Mb + 7.4 kb plasmid.
• Typical of reductive evolution, intracellular lifestyle, and parasitism.
• Many "essential" bacterial genes missing.
• Little evidence of horizontal gene transfer.
• High synteny between different serotypes.

Chlamydia Diagnosis

• PCR test is most sensitive, targeting a region of the C.pallidum genome.
• Non-synonymous mutations and pseudogenes differentiate strains causing different diseases.

Syphilis

Treponema Pallidum

• Three subspecies: T.pallidum subsp.pallidum (syphilis), T.p.subsp.endemicum (bejel or endemic syphilis), and T.p.subsp.pertenue (yaws).
• Genomes of all subspecies are ~1.14 Mbp.
• Limited metabolic capacity, many genes for transporters.
• Human-restricted pathogen, with humans as the only reservoir of T.pallidum.

Treponema Pallidum Genomics

• T.pallidum has dispensed with most biosynthetic machinery.
• No oxidative phosphorylation.
• No need for iron or cytochromes.
• Many transporters used to scavenge molecules from the host.

Diagnosis of Syphilis

• Detection mainly by serology, but inability to distinguish between subspecies.
• Methods:
  • Darkfield microscopy.
  • Direct fluorescent antibody staining.
  • Immunohistochemistry.
  • PCR becoming more common.
  • Point of care tests becoming more available.

Syphilis Treatment and Prevention

• Historically, treated with Salvarsan, the first synthetic antibacterial compound.
• Now, penicillin is the primary treatment.

GI Infections

• Gastrointestinal (GI) infections can be caused by bacterial, viral, or parasitic agents, with common symptoms including diarrhea, abdominal pain, vomiting, fever, nausea, and dehydration.
• Diarrheal diseases are a leading cause of morbidity and mortality, with 1.31 million deaths in 2017, primarily affecting children.
• The most common causes of diarrheal diseases are Rotavirus, Shigella, and Salmonella.

Shigella

• Shigella is a pathogenic biotype of E. coli, with four species that are considered serologically defined anaerogenic biotypes of E. coli.
• Shigella emerged from non-invasive E. coli through convergent evolution around 35,000-270,000 years ago.
• Despite its genetic similarity to E. coli, Shigella is still referred to by its original taxonomy due to its clinical significance.

Shigellosis: Disease and Transmission

• Shigellosis has a very low infectious dose (10-100 bacteria) and is acid-stable.
• The primary symptom of Shigellosis is diarrhea, which can range from watery to bloody (dysentery).
• Infection occurs in the colon and rectum, with clinical presentations ranging from asymptomatic to invasive.
• Shigellosis can be transmitted through person-to-person contact, contaminated food or water, and sexually.
• There were an estimated 188 million cases of Shigellosis in 2017, with approximately one-third occurring in Asia.

GI Infections

• Gastrointestinal (GI) infections can be caused by bacterial, viral, or parasitic agents, with common symptoms including diarrhea, abdominal pain, vomiting, fever, nausea, and dehydration.
• Diarrheal diseases are a leading cause of morbidity and mortality, with 1.31 million deaths in 2017, primarily affecting children.
• The most common causes of diarrheal diseases are Rotavirus, Shigella, and Salmonella.

Shigella

• Shigella is a pathogenic biotype of E. coli, with four species that are considered serologically defined anaerogenic biotypes of E. coli.
• Shigella emerged from non-invasive E. coli through convergent evolution around 35,000-270,000 years ago.
• Despite its genetic similarity to E. coli, Shigella is still referred to by its original taxonomy due to its clinical significance.

Shigellosis: Disease and Transmission

• Shigellosis has a very low infectious dose (10-100 bacteria) and is acid-stable.
• The primary symptom of Shigellosis is diarrhea, which can range from watery to bloody (dysentery).
• Infection occurs in the colon and rectum, with clinical presentations ranging from asymptomatic to invasive.
• Shigellosis can be transmitted through person-to-person contact, contaminated food or water, and sexually.
• There were an estimated 188 million cases of Shigellosis in 2017, with approximately one-third occurring in Asia.

Group A Streptococcus Overview

• Family: Streptococcaceae
• Genus: Streptococcus (medically important pathogens)
• Characteristics:
  • Gram-positive cocci
  • Facultative anaerobes
  • Non-motile
  • Host-adapted
  • Commensal of nasopharyngeal tract and skin
  • Opportunistic pathogen
  • Largely extracellular living
• Transmission: Aerosols and direct contact

Group A Streptococcus Characteristics

• Classified based on multiple factors
• Beta haemolytic
• Differentiated on basis of group carbohydrate (>12 serological forms)
• Group A carbohydrate: Polyrhamnose backbone + GlcNAc sidechain
• Catalase negative: Unable to convert H2O2 -> H2O + O2

Streptococcus pyogenes - Why Worry?

• Top 10 infectious disease agent
• Causes ~600,000 deaths/year
• S.pyogenes infections endemic in some areas
• Antibiotic resistance is a continual emerging problem
• No vaccine currently available

Streptococcus pyogenes - Infections

• Bacterial pharyngitis (Strep throat)
• Puerperal sepsis (Childbed fever)
• Necrotising fasciitis (Flesh-eating disease)
• Scarlet fever
• Infections: 700 million cases/year (superficial disease) and 1.78 million new cases/year (severe GAS disease)
• Deaths: >500,000 deaths/year

GAS causes Scarlet Fever

• Toxin-mediated disease primarily affecting young children
• GAS produces a variety of distinct toxins (superantigens like SpeA, SpeC, etc.)
• Superantigen expression leads to hyperstimulation of the host immune system

GAS Epidemiology

• 2011 Hong Kong scarlet fever outbreak
• Predominant GAS emm types: emm12 (>80%) and emm1 (>10%)
• Antibiotic resistance: Macrolide (>95%) and tetracycline (>80%) resistant

Genomics in GAS Epidemiology

• Importance of genomics in understanding bacterial evolution (e.g., outbreaks)
• Identifying virulence factors/mechanisms of pathogenesis
• Designing new vaccines and improving vaccine design
• Developing new antibiotics
• Developing new molecular diagnostics
• Designing public health interventions
• Monitoring pathogen populations' response

Learning from a Single Genome

• Complete genome sequence of an emm12 GAS from a scarlet fever patient
• Features: 1,908,100 bp, 1942 CDS, φ1 (46.4 kb), φ2 (45.1 kb), and ICE (64.9 kb)

Learning from Comparative Genomics

• Comparative analysis of HKU16 (emm12) and published non-scarlet fever emm12 genomes (MGAS9429 and MGAS2096)
• Identification of new prophages and ICE elements

The Genus Mycobacterium

• The genus Mycobacterium consists of 190 species, including major human pathogens such as M. tuberculosis and M. leprae.
• Mycobacteria are aerobic, rod-shaped bacteria that stain acid-fast using the Ziehl-Neelsen stain, and have a variable Gram stain.
• The cell wall of Mycobacteria is unique, with a thicker cell wall than other bacteria, composed of hydrophobic and waxy mycolic acids, and outer lipids linked to peptidoglycan by arabinogalactan.

Mycobacterial Lipids

• Mycobacterial lipids make up 30-60% of the cell's dry weight and are important for both structure and virulence.
• Many lipids are unique to each Mycobacterial species, and can activate or suppress the immune system, help survive inside macrophages, and evade the immune system.

Mycobacterium Tuberculosis

• M. tuberculosis is the causative agent of human tuberculosis, primarily infecting the lungs, but can disseminate to other tissues.
• It is a slow-growing, intracellular pathogen that divides every 18-24 hours, and takes 3-4 weeks to form colonies.
• M. tuberculosis has a complex lipids, including phthiocerol dimycocerosates (PDIMs), phenolic glycolipids (PGLs), and sulfolipids (SLs), which are important for virulence.

M. tuberculosis Virulence Factors

• PE/PPE family proteins are secreted by type VII secretion systems and comprise nearly 10% of the genomic coding potential.
• Five type VII secretion systems (ESX-1 to ESX-5) are involved in delayed phagosome maturation, phagosome rupture, and cellular necrosis.

Extrapulmonary TB

• Extrapulmonary TB occurs when M. tuberculosis disseminates outside of the lungs, and can occur in the pleural cavity, peritoneum, nervous system, genitourinary system, bones, and multiple sites simultaneously.
• It is more common in immunosuppressed individuals and HIV patients, and accounts for ~20% of all TB cases.

Tuberculosis Epidemiology

• In 2019, there were 10 million new cases of TB disease, with 1.5 million deaths, and 2 billion people with latent TB.
• The greatest disease burden is in low- and middle-income countries, with 10% of cases co-infected with HIV.

Origins of M. tuberculosis

• The origins of M. tuberculosis are still uncertain, but it is thought to have emerged from environmental mycobacteria through a stepwise adaptation to the intracellular environment.
• The "out of Africa" hypothesis suggests that the oldest relatives of M. tuberculosis are found exclusively in the Horn of Africa, and Africa has the largest diversity of M. tuberculosis lineages.

M. tuberculosis Vaccination

• The M. bovis BCG vaccine is the most widely used vaccine against TB, but it has highly variable protection and unknown duration of protection.
• There are 14 vaccine candidates in clinical trials, with the most promising being M72/AS01E, a recombinant fusion protein of two M. tuberculosis antigens, with ~50% efficacy in adult TB-positive populations.

M. tuberculosis Diagnosis and Treatment

• Microscopy and culture are still the main diagnostic tools for active disease, but new tools such as GeneXpert and line-probe assays are being developed.
• TB treatment typically involves multiple drugs given simultaneously over a long period, with 2 months of intensive treatment followed by 4 months of continuation treatment.
• Drug-resistant variants are emerging, with 480,000 cases of multidrug-resistant TB (MDR-TB) and 48,000 cases of extensively drug-resistant TB (XDR-TB) in 2018.

Non-Tuberculous Mycobacteria (NTM)

• Member of the Actinobacteria, with 190 species within the genus Mycobacterium, including major human pathogens M. tuberculosis and M. leprae.

Mycobacterium leprae

• Causative agent of Leprosy (Hansen's disease), a chronic infectious disease of nerves, skin, eyes, and nasal mucosa.
• Transmission is not completely defined, but believed to be spread by coughing or contact with nasal secretions from an infected individual.
• Epidemiology: >200,000 cases per year in >100 countries, with 80% of new cases in India, Indonesia, and Brazil.
• Armadillos, squirrels, and non-human primates are reservoirs.

M. leprae Pathogenesis

• Incubation period: 9 months to 20 years.
• Multiple forms of disease: tuberculoid (paucibacilliary) and lepromatous (multibacilliary).
• Tuberculoid: cell-mediated immune response limits M. leprae growth, with multiplication at site of entry and Schwann cell invasion.
• Lepromatous: impaired cell-mediated immune response, microbes proliferate within macrophages, and extensive penetration of M. leprae to distal sites.
• Treatment: dapsone + rifampicin + clofazimine for 6-12 months.

M. leprae Growth and Culture

• Very slow growing, with a doubling time of 12-14 days.
• Growth at 33°C, with tropism for peripheral nerves (lower temp) and Schwann cells and macrophages.
• Cannot be grown in the laboratory, and is an intracellular pathogen.
• Grown for study in 9-banded armadillos and footpads of athymic nude mice.

M. leprae Evolution/Genomics

• Highly reduced genome (3.3Mb).
• Opportunistic pathogen, seen in immunocompromised hosts.
• Increasing incidence over the last 20 years (~16/100,000).
• Treatment: 12-18 months of antibiotic therapy, with side effects common and treatment cost high.

Mycobacterium abcessus

• Emerging as a significant cause of NTM pulmonary disease in immunocompromised individuals.
• Produces biofilms, providing protection inside lungs of COPD/CF patients and protecting from drug treatment.
• Intracellular pathogen, surviving in macrophages.
• Causes skin infections, linked to acupuncture, tattooing, liposuction, and cosmetic surgery.
• Increasingly associated with cystic fibrosis patients, with 13% of CF patients having NTM infection.

Cutaneous NTM Infections

• Most frequently caused by M. marinum (SG), M. ulcerans (SG), M. chelonae (RG), M. abcessus (RG), and M. fortuitum (RG).
• High levels of antibiotic resistance, with water as the most common source of infection.

Mycobacterium marinum

• Causes "fish tank" granulomas, a pathogen of fish and humans, caused by direct contact of skin with contaminated source.
• Slow growing, with optimal temp of 30-32°C, and closely related to M. ulcerans.

Mycobacterium chelonae

• Often presents as disseminated disease, with multiple lesions, and can involve bone/joint infections.
• Mainly seen in immunosuppressed individuals, with high levels of resistance to standard anti-mycobacterial drugs.

Mycobacterium chimaera

• Associated with infection during heart bypass surgery, with risk of 50% mortality and difficult to treat.
• Slow growing, with no diagnostic tests, and a median incubation period of 17 months.

Anti-Mycobacterial Drugs

• Isoniazid: prodrug activated by mycobacterial enzyme KatG, binds tightly to InhA, and blocks fatty acid synthesis and mycolic acid synthesis.
• Ethambutol: inhibits arabinosyl transferase (embCAB operon), disrupting arabinogalactan biosynthesis and mycobacterial cell walls.

New Anti-Mycobacterial Drugs

• Bedaquiline: newest anti-TB drug (2012), diarylquinoline family, inhibits mycobacterial ATP synthase, and is bactericidal, active against replicating and dormant organisms.
• Delaminid/pretomanid: nitroimidazoles, inhibit cell wall synthesis (blocks mycolic acid synthesis), approved for MDR-TB.
• Telacebec (Q203): recently completed phase 2 (2020) for TB, targets cytochrome bc1, depleting ATP from cells, and highly active against M. tuberculosis.

Streptococcus pneumoniae

• First discovered in 1881 by Louis Pasteur, later identified by Carl Friedländer & Albert Fränkel (1884)
• Strictly human-adapted, no environmental reservoir
• Can reside asymptomatically in the upper respiratory tract
• Highly successful pathogen, estimated to cause 1.6 million deaths annually, one of the leading causes of mortality globally

Global Burden of Disease

• 1.6 million deaths annually, one of the leading causes of mortality globally
• USD$4 billion in costs (healthcare and loss of productivity) in the US alone
• WHO-designated priority pathogen, leading cause of community-acquired bacterial pneumonia (up to 50%)
• Ever-increasing rates of antibiotic resistance

S. pneumoniae Features

• Gram-positive, encapsulated, non-motile diplococcus
• Size: 0.5-1.5 µm
• Cell wall composed of:
  • Peptidoglycan
  • N-acetylglucosamine (GlcNac) and N-acetylmuramic acid (MurNac)
  • Teichoic acid (TA) and lipoteichoic acid (LTA)
• Most strains also encapsulated, capsule determines the serotype

Human Specialist

• Relatively small genome (2.0-2.1 Mbp), with little over 2000 genes
• Host-mediated genome decay
• Nutritionally fastidious, scavenges nutrients from the host wherever possible
• Auxotrophic for numerous amino acids, scavenges catalase by lysing host RBCs (α-haemolysis)
• Multiple incomplete biosynthetic pathways, e.g. glutathione
• Can adopt different 'lifestyles': carriage, disease

Pneumococcal Carriage

• Carried asymptomatically in the nasopharynx (~60% of children, ~5% of adults)
• Primary source of host-to-host transmission
• Carriage without dissemination often associated with specific serotypes
• Colonization is necessary for disease, followed by dissemination or invasion into deeper tissues

Pneumococcal Invasion

• Middle ear – otitis media: inflammation and pain in the middle ear, can result in perforated ear drums and deafness
• Lungs – pneumonia: inflammation of the respiratory tract and lung tissue, results in cough, shortness of breath, and fluid accumulation in lungs
• Blood – bacteraemia: also called blood poisoning, results in systemic inflammatory response
• Brain – meningitis: severe infection of the CNS, mortality rates up to 37%, long-term or permanent morbidity in 30-50% of surviving patients

Pneumococcal Pneumonia

• Responsible for > 1M deaths annually, ~7% of global deaths
• Greatest risk in the young and old
• Can be initiated by viral infection, requires antibiotic intervention

How to be a Successful Pathogen

• Pneumococcal capsule: extracellular polysaccharide that coats the bacterial cell wall
• Phase variation: random, high-frequency, reversible switching of gene expression
• Natural competence: ability to take up DNA from the environment
• Toxins (Pneumolysin): cholesterol-dependent cytolysins, binds to cholesterol on host cells, causing lysis

Capsule – What is it?

• Extracellular polysaccharide that coats the bacterial cell wall
• Made up of repeating sugar molecules (saccharides), poorly immunogenic
• Currently ~100 different serotypes, varying in sugar composition and linkage

Capsule – What does it do?

• Primary virulence determinant in S. pneumoniae
• Reduces mucociliary clearance, prevents complement deposition on the cell surface
• Facilitates survival in the blood, allows escape from NETs, and prevents opsonisation by antibodies

Therapeutics – Vaccines

• Pneumovax-23 (Merck): polysaccharide vaccine, recommended for adults
• Prevenar 13 (Pfizer): polysaccharide conjugate vaccine, given to infants
• Serotype replacement: emergence of new serotypes due to selective pressure of vaccination

Therapeutics – Antibiotics

• β-lactams (e.g. penicillin or amoxycillin), macrolides, tetracyclines, and cephalosporins are used
• Resistance reported up to 80% (!) in some countries
• Multi-drug resistance in up to 46% of isolates

Burkholderia Cenocepacia and Granulomatous Disease

• B. multivorans, Aspergillus, and Burkholderia are associated with granulomatous disease.
• B. cenocepacia is disproportionately associated with Cepacia syndrome, a necrotizing pneumonia.

Infections in Cystic Fibrosis Patients

• Bcc infections are common in cystic fibrosis (CF) patients, affecting approximately 5% of them.
• These infections are associated with high rates of morbidity and mortality in CF patients, with a higher mortality rate than P. aeruginosa.
• Bcc infections lead to enhanced rates of tissue damage, impacting lung function and making lung transplants necessary.

Burkholderia Pseudomallei and Melioidosis

• B. pseudomallei is a major cause of melioidosis, a disease with high mortality rates (up to 40% in some areas).
• Melioidosis is often underdiagnosed, making it essential to diagnose and start antimicrobial therapy early.
• B. pseudomallei infections can cause sepsis or localized abscesses in various organs, including the lungs, brain, soft tissue, and kidneys.

Pathogenesis Mechanisms

• Burkholderia species use various mechanisms to survive within hosts, including hiding PAMPs (pathogen-associated molecular patterns) and blocking the antimicrobial response.
• They employ secretion systems (type 3/6) to deliver effectors into host cells.
• Carbohydrates play a crucial role in pathogenesis, contributing to a protective barrier around the bacteria.

Glycosylation and Virulence

• Glycosylation is essential for virulence in Burkholderia species, with general glycosylation systems targeting multiple protein substrates.
• Flagellin glycosylation is observed in all pathogenic Burkholderia species, allowing them to evade immune recognition.
• General glycosylation leads to the installation of a conserved trisaccharide on proteins in the periplasm.

Immunogenic Properties of Glycans

• Protein glycosylation is required for virulence in Burkholderia species, but it is also highly immunogenic.
• Humans mount a robust humoral response to the Burkholderia glycan used for general glycosylation, which can be used to detect exposure to Burkholderia species.

Workshop 7: Identifying New Pathogens

Historical Context: 1976

• July 1976: Top hit record was "Kiss and Say Goodbye" by The Manhattans
• Football: VFL (not AFL) with no interstate teams, suburban grounds were used, and teams like Fitzroy and South Melbourne existed
• US President: Gerry Ford

Legionnaires' Disease Outbreak

• July 1976: Outbreak of a new disease at the State Convention of Legionnaires at the Bellevue-Stratford Hotel in Philadelphia, Pennsylvania, USA
• 182 cases of a disease spread via the respiratory tract
• 29 deaths (16% mortality rate)

Disease Identification

• December 1977: Papers published outlining the disease and the Gram-negative bacterium (Legionella pneumophila) that caused the disease
• Identification methods used: animal models, antisera, Gram stain

Investigation in 1976

• No genomics, no PCR, limited mass spectrometry
• If time-traveling with modern equipment, would need:
  • Equipment to investigate the outbreak
  • Consumables to pack into the Tardis