Infectious Disease Mechanisms

• Koch's third postulate is a starting point in understanding how disease agents cause disease.
• Determining the mechanisms involves:
  • Recapitulating the disease in an animal model
  • Measuring disease extent and severity
  • Identifying virulence factors
  • Defining steps in disease progression
  • Finding prevention and treatment strategies.
• Various animal models, from amoebas to primates, are used to investigate disease mechanisms.
• A suitable infection model system to measure virulence is critical for experiment design.
• The animal host must be susceptible to the pathogen and mirror disease symptoms of human infection as closely as possible.
• Accurately and reproducibly measuring virulence using the infection model is essential for identifying virulence factors and figuring out their roles.

Human Volunteers

• Human volunteers have been utilized in infectious disease studies
• Ethical considerations are essential, using human volunteers is more acceptable:
  • When the disease isn't life-threatening
  • Is easily treatable
  • Can be prevented with a vaccine
• Human volunteers have played a role in testing treatments and vaccines against:
  • Cholera
  • Chancroid (genital ulcers)
  • Gonorrhea
• These diseases lack suitable animal models
• Antibiotic intervention trials assessing the efficacy of antibiotic treatments are ethically defensible.
• Serious ethical questions have come about during someHuman Trials, such as the AZT trials in Thailand and Africa in the 1990s for prevention of mother-to-infant transmission of HIV.
• The control group received a placebo, whereas the trial group received a short-term course of AZT.
• This design would not be approved in developed countries.
• An ethically acceptable design in developed countries would compare the efficacy of a long course of AZT versus a shorter course of AZT.
• Scientists in developed countries found issues with the ethics of the short course trial, claiming that there was a different ethical standard in developing countries.

Tuskegee Syphilis Experiment

• The Tuskegee syphilis experiment was an unethical human trial conducted in the United States by the precursor of the NIH.
• Men with syphilis were not treated with antibiotics but observed as the disease progressed.
• The experiment was initiated in the 1930s and continued until 1972.
• In 1932, the U.S. Public Health Service in Alabama decided to withhold treatment from 399 African-American men with syphilis.
• The goal was to learn more about the development and pathology of the disease.
• The men were offered "free health care," consisting of regular clinic visits, detailed records, and treatment for ailments other than syphilis.
• When penicillin became available in 1947 as an effective and nontoxic cure for syphilis, therapy was still withheld.
• By 1972, 28 of the men had died of syphilis, and 100 others had died of syphilis-related complications.
• The study was designed and carried out by people working for public health agencies, not by Tuskegee University.
• The experiment provoked congressional hearings, which formed the basis for current ethical rules governing the use of human volunteers.
• The U.S. government offered free medical care to the men involved and later extended it to family members, including infected wives and children.
• A lawsuit was required to prompt the government to offer a cash settlement.
• Public Health Service officials defended the study, arguing that the men would not have had treatment anyway.
• It resurfaced in connection with the controversial AZT trial design because it was unethical research and governmental lack of concern.
• Critics of the AZT study protested that different standards of medicine were being applied to people in the developing world.
• The examples described eventually led to the current ethical guidelines and federal regulations concerning human subjects research.

Belmont Report

• The Belmont Report of 1979 identified three ethical principles:
  • Respect for persons (informed consent)
  • Beneficence (maximize benefit, minimize harm)
  • Justice (fair treatment and risk distribution among potential participants)
• The Common Rule was established in 1981 as a baseline standard of ethics governing biomedical and behavioral research involving human subjects
• Research must be approved in advance by Institutional Review Boards (IRBs)
• IRBs consist of experts, including physicians, statisticians, and bioethicists, and enforce regulations established by the NIH and other funding agencies.

Prospective Studies

• A clinical trial study involving inoculation of humans with a disease agent or treatment with a new drug/vaccine is called a "prospective" study.
• A prospective study follows a cohort of human subjects, who may differ with regard to certain traits, exposures, or treatments.
• Differences in these traits, exposures, or treatments may impact disease outcome.
• An example of a prospective study is one that studies the role of bacterial vaginosis in pregnant women as a risk factor for preterm birth.

Animals and Measurements of Virulence

• Animal models, such as mice, rats, guinea pigs, rabbits, dogs, primates, and pigs, have expanded as tools in research have improved.
• Smaller, less expensive animals, like:
  • Fruit flies
  • Nematodes
  • Amoebas
• Smaller animals can be infected in large numbers, facilitating high-throughput screens for virulence factors.
• The route of infection affects virulence.
• Bacteria delivered intravenously can access different tissues than those delivered intranasally.
• Physiological differences between animal models and humans must be considered.
• Virulence measurement include:
  • Body weight loss
  • Fever
  • Internal bacterial counts
  • Survival curves
  • Colonization rates
  • Pathology caused by bacteria
• The goal is to find an animal model that closely mimics the human infection process.
• Colonization of a mucosal tissue surface is necessary before infection can occur and is often studied.
• Mutants defective in colonization are useful for identifying bacterial colonization factors.
• Dissemination studies track pathogen spread using fluorescence or bioluminescence.
• Pathogenicity assays reveal the impact of disease on an organism's health through observation or measurement.
• Virulence can be measured through LD50 (lethal dose 50) and ID50 (infectious dose 50) values.
• The LD50 is the number of bacteria needed to kill 50% of infected animals.
• The ID50 is the number of bacteria needed to cause clinical signs of infection in 50% of infected animals.
• Lower LD50 or ID50 values indicate higher virulence.
• Calculating the ratios of the number of CFU recovered from the infected host provides a means to measure how well the bacteria can replicate in the host
• Determining CFU numbers requires sacrificing test animals at different time points, leading to the development of rapid imaging techniques.
• Two techniques developed are:
  • Bioluminescent imaging
  • Fluorescent imaging
• The infecting bacteria are made to express luciferase, which produces light, or fluorescent proteins, which can be detected by appropriate filters.
• Luminescence from luciferase-expressing bacteria must be distinguished from animal autofluorescence by software.
• Mutant studies that affect virulence need to be conducted carefully as it can negatively impact growth.

Competition Assays

• Competition assays are used to measure bacterial virulence.
• In competition assays, an animal is infected with a mixture of the parent bacterial strain and a mutant strain.
• The mutant has a marker so that the parent and mutant can be distinguished.
• If the mutation strongly affects virulence, then the mutant population will decrease compared to the wild-type.
• A virulence assay was used where a marked mutant of S. Typhimurium was introduced into mice at the same time as the wild-type strain.
• If the Himar mutation caused an increase in colonization and survival in the host, then the mutation caused an increase in colonization and survival in the host.
• Competition assays compare the fitness of the mutant to compete with the wild-type bacterium for survival in the host environment.
• It is often easier to quantify the ratio of the wild type to mutant bacteria more accurately than it is to determine the ID50 or LD50 value accurately.
• Competition assays allow investigators to use far fewer animals, since it is not necessary to use multiple animals for each dose tested to generate a dose curve.
• Mutants that grow more slowly in broth cultures will likely lose in competition assays in an animal, so this does not mean that the process affected in the mutant is necessarily contributing directly to the virulence process.
• It can also be difficult to interpret what subtle differences obtained in competition experiments actually mean in terms of the mechanism of virulence.
• The competition experiment paradigm precludes the use of some detection approaches, such as biophotonic imaging, because the light produced from the parent or mutant is indistinguishable.
• A serious concern is trans-effects that may allow mutants defective in virulence to grow in the presence of the wild-type parent strain.
• Usually a combination of single-strain and competition infection experiments is needed to begin to understand processes as complicated as bacterial virulence in animal models.

Tissue Culture

• Animal models are the "gold standard" of research on bacterial virulence.
• Animals present a complex system in which many variables cannot be controlled.
• Cultured mammalian cells are commonly used to provide a more easily controlled system for investigating host-bacterium interactions.
• Tissue culture cells can be grown in defined medium under reproducible conditions with only one or a limited number of cell types represented, which makes measurements and interpretations easier and more reproducible.
• It is also easier to perform experiments involving radioactive compounds and to introduce foreign DNA into tissue culture cells.
• Cultured cells can be readily visualized by microscopic techniques, and cells expressing certain fluorescently labeled marker proteins can be sorted using high-speed devices.
• They also cost less per day to house than laboratory rodents, do not fight with each other, and have seldom been known to escape from their cages.
• Because of the very important role that tissue culture cells have played in molecular investigations of bacterium-host cell interactions, it is important to understand their limitations, which must be kept in mind when interpreting results of experiments.
• Primary cultures of mammalian cells that are not derived from tumors can be obtained from animal tissue, biopsy material, or the blood of human volunteers (e.g., macrophages)
• Primary cell types usually undergo a limited number of divisions in culture and can only be maintained for a couple of weeks.
• Consequently, "immortalized" cells that continue to divide in culture are often used in routine experiments.
• These immortalized cells are derived directly from a tumor, by fusing a primary cell type with a tumor cell, or by continuous selection of primary cell lines for unregulated growth.
• Propagation of mammalian cells that are unregulated for growth in culture leads to the accumulation of numerous mutations, gene rearrangements, and gene duplications.
• Mutations are not only uncharacterized because they are so numerous and complex, but they are also not easily reproduced.
• Two separate sets of primary cells from the same source that are treated in exactly the same way to produce immortalized cell lines will have different combinations of mutations and rearrangements.
• Another way of immortalizing the cells is to introduce certain viral proteins into them.
• Cultured cells are far from being genetically identical to cells in the tissue or organ from which they were derived.
• Different lines developed from the same type of cell are not necessarily identical.
• Cell lines that are passed repeatedly in culture will continue to develop new mutations.
• Cells in culture are no longer in the same environment as in the organ of origin, and many genes that were expressed by cells in an intact organ may not be expressed in cultured cells.
• Culture medium does not represent the natural conditions and biochemical composition found in the body, where fluids and cellular secretions may significantly influence cellular processes in ways that are not reproducible in a culture system.
• Tissue culture cell lines lose many traits of the original tissue from which they were derived and may not display the same surface markers in culture.
• Bacterial and viral pathogens that are highly specific for a particular tissue or cell when causing an infection in an intact animal are frequently able to invade cultured cells derived from tissue they do not normally infect.
• Differences in gene expression can be corrected to some extent by addition of growth stimulants or hormones to the culture medium or by providing an artificial matrix (also called a substrate) for the cells to grow on.
• Most cultured cells lose their normal shape and distribution of surface antigens.
• Cells in an intact animal are usually polarized, meaning that different regions of the cell surface membrane are exposed to different environments.
• Membranes on different sides of polarized cells contain different sets of proteins, a feature that is presumably important for their function.
• In a layer of normal mucosal cells, the apical surface is exposed to the external environment, whereas the basal and lateral surfaces are in contact with the extracellular matrix.
• Mucosal cells in the gastrointestinal tract and some other tissues are also connected to each other by tight junctions.
• Tissue culture cells that are grown as nonconfluent monolayers do not have differentiated surfaces.
• Proteins that are found only on the apical or on the basal-lateral surfaces of cells in vivo may be distributed over the entire surface of such cells, assuming they are produced at all.
• Allowing the cells to grow to confluence does not necessarily solve this problem, and it is usually necessary to provide an extracellular-matrix substitute and to provide hormones to obtain a polarized monolayer in culture.
• Human mucosal surfaces are covered with mucus and bathed in solutions that are difficult to mimic in an in vitro system.
• The fact that there are problems with existing tissue culture cell lines does not mean that such cell lines are not extremely useful and can be marvelous tools for discovery if their limitations are kept in mind.
• In general, once a new phenomenon has been discovered in cultured cells (e.g., attachment of bacteria to a mammalian cell receptor or reorganization of host cell cytoskeleton), experiments can be designed that use organ cultures or animals to test the importance of the phenomenon in vivo.
• It is not uncommon for a mutation that affects the ability of a bacterium to infect a tissue culture cell to have no effect when tested in animals, so taking the study from tissue culture cells to the animal is an important step.
• Tissue culture cells can be important for generating hypotheses that need to be tested in intact animal models.
• It is often difficult, however, to take the results obtained from studies of tissue culture cells and extrapolate them directly to the disease in humans.

Gentamicin Protection Assay

• Tissue culture cells have been widely used to study adherence properties of bacteria and are particularly useful for identifying virulence factors involved in binding and invasion of host cells by intracellular pathogens.
• The gentamicin protection assay distinguishes between mutants that are defective in adhesion (attachment to the cell surface) from those that are defective in invasion.
• A culture (or monolayer) of mammalian cells is incubated with bacterial cells at a certain multiplicity of infection (MOI), which is defined as the number of input bacterial cells per mammalian cell that are present in the culture tube or well of a culture plate.
• Following incubation for a period of time to allow binding and invasion to occur, the samples are divided into three tubes or plate wells.
• The first tube provides the total number of bacteria present (CFUs) in the tube (or well) of mammalian cells.
• The pellet is resuspended in buffer with mild detergent or with gentle scraping to lyse the mammalian cells.
• The resulting suspension of bacteria is serially diluted and plated on agar plates, and the bacteria are allowed to grow to form colonies, which are counted.
• The second tube provides the total number of adherent (attached) bacteria (CFUs).
• The infected mammalian cells are centrifuged and the pellet is first washed with a buffered solution several times to remove any nonadherent (unattached) bacteria.
• The mammalian cells with adherent bacteria are then lysed, and serial dilutions of the suspension are plated to determine the number of cell-associated bacteria (denoted as adherent CFUs).
• The ratio of cell-associated CFUs to total CFUs at the end of the experiment is defined as the adhesion frequency.
• A third tube is centrifuged and washed to remove nonadherent bacteria, and fresh culture medium containing the antibiotic gentamicin is added to the tube.
• Gentamicin cannot enter mammalian cells, so it kills only extracellular bacteria and does not kill bacteria that have already entered the mammalian cells.
• The cells are incubated with the gentamicin-containing medium for anywhere from 30 minutes to 2 hours, at which time the culture is centrifuged and the medium is removed.
• The mammalian cells are washed to remove unattached bacteria and mammalian cells are lysed and the bacteria resuspended, as described previously.
• Serial dilutions of the bacterial suspensions are then plated to determine the number of intracellular bacteria (denoted as gentamicin-resistant CFUs).
• The ratio of gentamicin-resistant CFUs to adherent CFUs at the end of the experiment is defined as the invasion frequency.
• It is possible to report the number of gentamicin-resistant CFUs to total number of CFUs in the well, which may be very different depending on the frequency of adherence and growth rate of a given bacterial strain for the given mammalian cells used.
• If a bacterial mutant is defective in an adhesion factor that prevents it from attaching to the mammalian cell, then no colonies will form on the agar plate for the second and third sets of tubes or wells.
• Mutants that can still adhere but are defective in invasion factors, so that they cannot enter mammalian cells or survive once inside, will produce colonies on plates from the second set of tubes or wells, but will have no colonies on plates from the third set of tubes or wells.
• By comparing the adherence frequencies and the invasion frequencies for wild-type bacteria versus mutants, it is possible to determine the nature of the virulence factor that was defective in the mutant bacteria.

Invasion Success Curve

• Studying pathogenic mechanisms of Campylobacter jejuni, a foodborne pathogen that causes diarrhea, is hampered by the lack of simple animal models that mimic human disease, so cell culture assays have provided useful alternative ways to investigate C. jejuni interaction with host epithelial cells.
• Use of the gentamicin assay has shown that invasiveness varies considerably depending on the C. jejuni strain, as well as the human cell lines used, the number of bacteria in the inoculum (MOI), and other assay conditions.
• Researchers in the field have sought to standardize assay conditions and to set up tests for comparisons between strains.
• One such test is to generate an invasion success curve, which plots the log(MOI/cell) versus the log(invaded bacteria/cell) to give the minimal MOI (minMOI) required to obtain the maximum number of internalized bacteria per mammalian cell (BImax).
• In comparing strains, those with lower minMOIs are more invasive.

Plaque Assay

• Some intracellular pathogens, such as Legionella and Chlamydia, are able to invade eukaryotic cells, multiply within the eukaryotic cell until they burst from the host cell, and then spread from one host cell to the adjacent host cell.
• Bacteria replicate within each invaded host cell, killing the host cell in the process and creating a cleared zone of killed cells (called a plaque) around the initial cell that was invaded.
• Some intracellular pathogens, such as Listeria and Shigella, are also able to spread laterally within a monolayer by propelling themselves into the adjoining cell without being released into the medium.
• A modified gentamicin tissue culture assay that allows for assessment of an intracellular pathogen’s ability to spread from cell to cell is the plaque assay.
• Tissue culture cells are grown on plates to form an even confluent monolayer and bacteria are added to the medium and incubated with the mammalian cells for a short period of time to allow invasion to occur.
• The medium with unattached bacteria is removed and the monolayer is incubated with gentamicin to kill any remaining extracellular bacteria.
• The monolayer is then gently covered with another layer of soft agar containing gentamicin (to prevent diffusion of the bacteria through the medium) and the cells are further incubated.
• The living cells are stained and plaques (cleared areas) in the monolayer can be observed where bacteria have invaded and spread from cell to cell, killing the mammalian cells in the process.
• Mutants that are defective in efficient cell-to-cell motility will form small plaques, while mutants that are defective in factors necessary for invasion and/or intracellular survival or replication do not form any plaques.

Fluorescence Microscopy Techniques

• The development of new reagents for probing the interior of a mammalian cell or for changing its chemical environment has generated opportunities for sophisticated new approaches to study bacterium-host cell interaction.
• Fluorescent dyes attached to specific reagents (chemicals, proteins, or monoclonal antibodies) that bind specifically to host cell cytoskeletal components, such as actin, have been used to follow by fluorescence microscopy the cytoskeletal rearrangements caused by bacteria attaching to and invading host cells.
• These methods have also been used to observe the changes in cellular morphology caused by toxins or secreted effector proteins produced by bacteria.
• One particularly useful reagent for visualization of cytoskeletal rearrangements caused by the formation of actin filaments in cells is the marine metabolite phalloidin
• Phalloidin is a compound that binds very tightly to polymerized F-actin (which forms the actin filaments), but not free monomeric G-actin.
• When phalloidin is linked to a fluorescent dye, such as rhodamine (red) or fluorescein isothiocyanate (green), fluorescence microscopy can be used to monitor actin cytoskeletal changes and actin stress fiber formation.
• Calcium release from intracellular stores (called calcium mobilization) is another intracellular response often triggered by interactions of a cell with bacteria or bacterial toxins.
• A number of fluorescent dye reagents (such as Fura-2) that can detect calcium levels inside tissue culture cells are available.
• Cellular markers are mammalian proteins that are known to localize to particular compartments within the host cell.
• They are introduced into the mammalian cells by transfection with mammalian expression vectors encoding genes for these cellular marker proteins that have been modified with tags, such as green or red fluorescent proteins or epitope tags, which are recognized by antibodies conjugated to fluorescent dyes.
• Robotically controlled, high-throughput fluorescence microscopes are now available that allow rapid screening for changes in the infected host caused by bacterial mutants.

Organ Culture Models

• Organs or portions of them can now be kept viable in vitro for longer and longer periods of time
• In the case of organ cultures, techniques for in situ quantitative detection of proteins and mRNA make it possible to detect elevated expression of bacterial genes in a particular tissue.
• An advantage of using organ cultures is that there are usually multiple cell types present, including some of the immune system, which might allow a better approximation of what is happening during a natural infection.
• Organ cultures provide a much better model than tissue culture cells for what transpires in an animal or a human host, but they may be more difficult to obtain and maintain.
• An organ culture can begin to deteriorate within hours or days, making it difficult to do long-term experiments.
• One example is the organ culture model for the foodborne pathogen C. jejuni, in which researchers have used human gastrointestinal tissue obtained from endoscopic biopsies of patients to highlight the surprising propensity of C. jejuni to adhere to mucosal tissue via its flagellum rather than pili, which are more typically used by bacteria for surface adhesion.
• Scientists have benfitted from the popularity of cosmetic surgery and hysterectomies.
• Scientists have begun to develop artificial organ cultures
• One example of this is artificial skin equivalents, which are made by culturing a suspension of human foreskin fibroblasts in a matrix of native acid-soluble collagen and serum-supplemented medium at 37°C, where the collagen polymerizes and traps the cells, which then elongate and spread for several days.
• Freshly isolated suspensions and connective-tissue can then allow better adhesion to the suspension.
• Some of the more exciting recent advances in the area of organ culture research have been in the development of multiorgan microphysiological systems that can serve as human surrogates for drug screening and testing.
• These microscale platforms enable organ-organ interactions in cell cocultures using microfluidic devices, where the medium connects the organ tissues through diffusion or circulation.

The Continuing Need for Reliable and Plentiful Information about Disease Pathology

• A topic that is seldom discussed, but is essential for making progress in pathogenesis research, is the quality and quantity of information available about how a disease progresses and, perhaps even more important, the extent to which basic scientists in the field are aware of this information.
• To completely understand the complex nature of host-pathogen interaction and its disease outcome, it is extremely important for researchers to acquire key knowledge of human physiology or the physiology of whatever animal they are using as a model.
• One must know as much as possible about the normal distribution of the bacteria in the body during infection, and about how and what type of damage occurs as the disease progresses.
• When the physiological relevance is taken into consideration, the results of experiments on tissue culture cells and other ex vivo model systems are more likely to be interpreted correctly and to provide new insights that could lead to improved treatments.