How can a pathogenic bacterium be avirulent?

How can a pathogenic bacterium be avirulent?

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Streptococcus pneumoniae R6 is a pathogenic bacterial strain but it is avirulent. How can a pathogen be avirulent. What does it mean if a pathogenic bacterium is avirulent?

Streptococcus pneumoniae is a pathogenic species. Streptococcus pneumoniae R6 is an avirulent strain of this species. Presumably this means that specific virulence genes that are present in virulent strains of Streptococcus pneumoniae are missing or mutated in the R6 strain. In fact the most likely difference would be that virulence factors are encoded by a plasmid that has been lost in the avirulent strain.

The use of avirulent strains is a common strategy for studying pathogenic bacteria, although obviously it precludes studying mechanisms of pathogenicity directly.

Avery, MacLeod, and McCarty Identified DNA as the Genetic Material

3.2.4 Griffith’s Decisive Experiment: The Discovery of Bacterial Transformation

Griffith noticed that while some serotypes of stable R variants of pneumococcus did not revert in the mouse into encapsulated S forms, revertants were detected when the R cells were coinoculated with heat-killed S bacteria. Expanding on these preliminary observations, he defined experimental conditions that maximized the capacity of heat-killed S cells to affect the transformation of R cells into virulent encapsulated S pneumococci. In these preparatory experiments Griffith identified combination of serotypes of the heat-killed S cells and living R bacteria that yielded significant R to S transformation in the mouse. Thus, for instance, R cells that were derived from Type SII cells were successfully transformed when they were coinjected with heat-killed SI bacteria. Griffith also defined optimal temperature, usually 60°C that effectively killed the S bacteria while allowing for significant transformation. He also found that transformation occurred only when the inoculum size of the heat-killed S cells was much larger than that of the living R cells. Importantly, using heat-killed S cells and alive R cells of two different serotypes, Griffith noticed that under proper conditions the R to S transformed cells acquired the serotype of the heat-killed S cells rather than maintaining the serotype of the original R cells. 37 Next, Griffith conducted fully controlled experiments that are schematically depicted in Fig. 3.8 . Multiple experiments (see Fig. 3.9B for representative original results) showed that neither R cells nor heat-killed S pneumococci by themselves could produce disease and death in mice. By contrast, injected R cells that were mixed with a large excess of heat-killed S bacteria caused death in the mice. The serotype of bacteria that were recovered from the dead animals was that of the heat-killed S cells and not the different original serotype of the R cells. Typical features of the transformation of pneumococcus in Griffith’s hands (and in the hands of researchers who later replicated his results) are shown in Fig. 3.9B .

Figure 3.8 . Scheme of the Griffith transformation experiment. In control experiments mice that were infected with encapsulated virulent Type S pneumococci (serotype I) died and SI bacteria were recovered from the dead animals. By contrast, injected heat-killed SI bacteria did not cause death and no bacteria were detected in sacrificed animals. Similarly, capsule-less R bacteria (serotype II) did not cause death and no bacteria were detected in sacrificed mice. By contrast, mice that were co-inoculated with mixture of RII pneumococci and of large excess of heat-killed SI cells did die and the bacteria that were recovered from the dead animals were serotype I of the heat-killed S cells.

Figure 3.9 . Results of typical transformation experiments. (A) Scheme of successful transformation by mixture of living RII pneumococci and heat-killed SI bacteria. (B) Summary of the actual results (Table VII from Griffith F. The significance of pneumococcal types. J Hyg (Lond) 192827(2):113–59. Control mice that were injected with only heat-killed SI bacteria and remained unaffected are encircled in black. Successfully transformed colonies are encircled in red. Red asterisks mark untransformed R cells that were recovered from sacrificed unaffected mice in cases of ineffective transformation.

First, whereas heat-killed S cells alone did not produce disease and death in mice, injection of their mixture with living R cells resulted in some, but not all, cases in death of the animals.

Importantly, the newly encapsulated transformed bacteria that were recovered from the hearts of infected dead mice were of serotype I of the heat-killed S cells and not of Type II from which the R cells were originally derived. Notably, Griffith also executed the reverse experiment, that is, mice that were inoculated with a mixture of heat-killed SI cells and living RII pneumococci died and produced living SI cells.

Two features of the results shown in Fig. 3.9B are noteworthy. First, transformation did not always occur. Rather, whereas it was observed in some cases (mice 645 and 650 in Fig. 3.9B ), in other instances the mice remained asymptomatic and the sacrificed animals either had no detectable bacteria (mouse 651) or they bore some untransformed R pneumococci (mice 646–648 and 652). Second, to affect even this inconsistent transformation, the heat-killed S cells had to be added at an excess of about 200-fold over living R bacteria.

Although the source for the incomplete efficiency of transformation and for the need for a large excess of heat-killed S cells was unclear at the time, these features are easily understood in hindsight. As will be discussed later, transformation of pneumococci by DNA was inefficient, especially under the less than optimal conditions that were employed by Griffith and his immediate successors. It was impossible, therefore, to obtain uniformly successful transformation even when heat-killed S cells were added at a great excess over R cells. Significantly, Griffith also tried to achieve transformation in vitro by mixing in the test tube heat-killed S cells with living R bacteria. However, neither R to S conversion nor transformation of serotype was attained. This failure to achieve transformation in vitro was most likely due to experimental conditions that were suboptimal for efficient transformation and to degradation of the transforming material by enzymes that were released from autolyzed R cells. 40

How can evolutionary biology help to get rid of antibiotic resistant bacteria?

The evolution of antibiotic resistance. Credit: Shutterstock

Craig MacLean, Professor of Evolution and Microbiology at Oxford's Department of Zoology, explains how evolutionary biology can help us to get rid of antibiotic resistant bacteria.

Bacteria are tiny single cell organisms, invisible to the naked eye, that live in essentially every possible habitat on our planet. Plants and animals are covered with microorganisms, the soil and the oceans are teaming with bacteria, and it is estimated that bacterial cells actually outnumber human cells in the body by a factor of 10-100:1. The overwhelming majority of bacteria are completely harmless, but a small minority of pathogenic bacteria can cause infections in humans. For most of human history, bacterial pathogens have been a major cause of disease and mortality. For example, the plagues that ravaged Europe in the middle ages were caused by the bacterium Yersinia pestis, and tuberculosis, and cholera outbreaks are caused by the bacterium Vibrio cholera.

The development of antibiotics in the 1940s provided a simple and effective treatment for many bacterial infections for example, antibiotics decreased the mortality rate associated with serious cases of pneumonia from 90% to 10%. Given these stunning results, many prominent members of the medical community, including the US Surgeon General, thought that antibiotics would effectively make bacterial disease a thing of the past. Against this background of boundless optimism, researchers had already discovered that bacteria could become resistant to antibiotics and Alexander Flemming, who led the team that discovered penicillin, warned that the misuse of antibiotics would lead to the rise of resistance, rendering antibiotics ineffective.

Antibiotics have now saved millions of lives, but the large-scale use of antibiotics has driven the spread of resistance, as predicted by Flemming. Pathogenic bacteria have evolved resistance to all of the main classes of antibiotics and pan resistant bacteria have caused untreatable infections. Resistance already imposes a substantial health and economic burden, and an influential report published by the O'Neill commission in 2016 predicted that resistant infections could cause 10 million deaths per year and impose a global financial cost of 100 Trillion USD by 2050 .Given this threat, resistance has been identified as one of the most important global challenges by organisations such as the United Nations, The G8 and even the International Monetary Fund.

The spread of antibiotic resistance in pathogenic bacteria is a simple and elegant example of evolutionary adaptation by natural selection. Bacteria can become resistant to antibiotics through mutations that alter the cellular targets of antibiotics or by acquiring dedicated resistance genes from other bacteria. The acquisition of resistance is a very rare event for example, resistance mutations usually occur in less than 1 in a million bacteria. However, resistant bacteria can continue to grow and reproduce under antibiotic treatments that effectively paralyse or kill their antibiotic susceptible neighbours—this is Darwinian natural selection in its simplest and cruellest form. Rare resistant strains can rapidly come to dominate pathogen populations under antibiotic treatment, and, in a worst-case scenario, these resistant bacteria can then go on to infect other people.

This simple sketch shows how evolution drives the spread of resistance, but it leaves out many important details. Evolutionary biologists and microbiologists have become increasingly interested in understanding the processes driving the spread and maintenance of resistance. These studies have addressed a wide-range of important questions, such as: What limits the transmission of resistant bacteria between people? How does the strength of antibiotic treatment influence the likelihood of resistance emerging? Can antibiotic cocktails be used to suppress the evolutionary advantage of resistance? How do resistance genes move between bacteria? We now have quite a mature theoretical framework for thinking about these important questions. The problem, however, is that the largely theoretical approach the evolutionary biologists have taken to resistance is not very well connected to the reality of resistance in the clinic.

In the last 15 years, technological innovations have massively improved our ability to sequence the genetic code of all living organisms, especially bacteria. Sequencing the genomes of pathogenic bacteria isolated from infections has provided a much clearer picture of how resistance emerges and spreads, especially in hospitals. In many important human pathogens, the global increase in the prevalence of antibiotic resistance has been driven by the epidemic spread of a relatively small number of highly resistant 'superbugs' that transmit between people, such as MRSA Staphylococcus aureus and XDR Mycobacterium tuberculosis. If evolutionary biology is going to help contribute to stopping the spread of antibiotics resistance, the field is going to need to shift emphasis towards understanding the specific processes that have driven the emergence of the superbugs.

For more information, read: "The evolution of antibiotic resistance" in Science.

Primary Pathogens versus Opportunistic Pathogens

Pathogens can be classified as either primary pathogens or opportunistic pathogens. A primary pathogen can cause disease in a host regardless of the host’s resident microbiota or immune system. An opportunistic pathogen, by contrast, can only cause disease in situations that compromise the host’s defenses, such as the body’s protective barriers, immune system, or normal microbiota. Individuals susceptible to opportunistic infections include the very young, the elderly, women who are pregnant, patients undergoing chemotherapy, people with immunodeficiencies (such as acquired immunodeficiency syndrome [AIDS]), patients who are recovering from surgery, and those who have had a breach of protective barriers (such as a severe wound or burn).

An example of a primary pathogen is enterohemorrhagic E. coli (EHEC), which produces a virulence factor known as Shiga toxin. This toxin inhibits protein synthesis, leading to severe and bloody diarrhea, inflammation, and renal failure, even in patients with healthy immune systems. Staphylococcus epidermidis, on the other hand, is an opportunistic pathogen that is among the most frequent causes of nosocomial disease. [2] S. epidermidis is a member of the normal microbiota of the skin, where it is generally avirulent. However, in hospitals, it can also grow in biofilms that form on catheters, implants, or other devices that are inserted into the body during surgical procedures. Once inside the body, S. epidermidis can cause serious infections such as endocarditis, and it produces virulence factors that promote the persistence of such infections.

Other members of the normal microbiota can also cause opportunistic infections under certain conditions. This often occurs when microbes that reside harmlessly in one body location end up in a different body system, where they cause disease. For example, E. coli normally found in the large intestine can cause a urinary tract infection if it enters the bladder. This is the leading cause of urinary tract infections among women.

Members of the normal microbiota may also cause disease when a shift in the environment of the body leads to overgrowth of a particular microorganism. For example, the yeast Candida is part of the normal microbiota of the skin, mouth, intestine, and vagina, but its population is kept in check by other organisms of the microbiota. If an individual is taking antibacterial medications, however, bacteria that would normally inhibit the growth of Candida can be killed off, leading to a sudden growth in the population of Candida, which is not affected by antibacterial medications because it is a fungus. An overgrowth of Candida can manifest as oral thrush (growth on mouth, throat, and tongue), a vaginal yeast infection, or cutaneous candidiasis. Other scenarios can also provide opportunities for Candida infections. Untreated diabetes can result in a high concentration of glucose in the saliva, which provides an optimal environment for the growth of Candida, resulting in thrush. Immunodeficiencies such as those seen in patients with HIV, AIDS, and cancer also lead to higher incidence of thrush. Vaginal yeast infections can result from decreases in estrogen levels during the menstruation or menopause. The amount of glycogen available to lactobacilli in the vagina is controlled by levels of estrogen when estrogen levels are low, lactobacilli produce less lactic acid. The resultant increase in vaginal pH allows overgrowth of Candida in the vagina.

Think about It

  • Explain the difference between a primary pathogen and an opportunistic pathogen.
  • Describe some conditions under which an opportunistic infection can occur.

How to Design Vaccines? | Biology

Vaccines may be defined as a preparation of antigenic materials, often combined with adjuvants, that is adminis­tered to individuals to induce protective immunity against pathogenic infections.

The antigen may be in the form of live but avirulent microorganisms, killed micro­organisms, purified macromolecular antigenic component of a microorganism, or a plasmid that contains a complementary DNA encoding a microbial antigen.

Most commonly used vaccines work by inducing humoral immunity, and attempts to stimulate cell-mediated immune responses by vaccination are ongoing.

The success of active immunization with vaccines in eradicating infectious disease is dependent on several factors:

1. Vaccines must be able to recognise the important differences between activation of the humoral and cell-mediated immune responses.

2. Vaccines should be able to develop immunological memory.

3. Vaccines should be most effective against infections that are limited to human hosts and are caused by poorly infectious agents whose antigens are relatively invariant.

Approaches to Design Vaccines:

1. Whole organism vaccine:

Many com­monly used vaccines consist of:

(ii) Live but attenuated (avirulent) bacterial cells or viral particles.

(i) Attenuated viral and bacterial vaccines:

In such cases, microbes are treated in such a way that they can no longer cause disease (i.e., their virulence is attenuated or weakened) but retain their capacity for transient growth within an inoculated host.

Attenuation can be achieved by growing the microbes for prolonged periods under abnormal culture conditions. Such process selects only the mutants that can grow better in abnormal culture but are less capable of growth in normal host.

Vaccines of tuberculosis was developed from attenuated strains of Mycobacterium bovis called Bacillus Calmette-Guerin (BCG) rubella vaccines from attenuated strain of rubella virus and Sabin polio vaccine developed from attenuated viral strain, are common examples of such vaccines.

(1) Such vaccines result in increased immunogenicity and production of memory cells as a result of which these vaccines often require only a single immu­nisation and there is no need for repeated boosters.

(2) Such vaccines often induce long- lasting specific immunity, so immunisation in childhood is sufficient for lifelong protection.

(1) The main disadvan­tages of such vaccines are the possibility to reversion in a virulent form from the attenuated state of the microbes.

(2) Presence of other viruses as contaminants may cause some adverse reactions to the recipients.

(3) Sometimes post-vaccine complications may render a potential vaccine unacceptable.

(ii) Inactivated viral or bacterial vaccines:

A common approach in vaccine production is the use of inactive microbes. In this case, the microbes are inactivated by heat or by chemical means so that they are no longer capable of replication in the host but the structure of epitopes are maintained. Chemical inactivation by formaldehyde or other alkylating agents are more successful than heat treatment.

Whooping cough vaccine, Salk polio vaccine, HIV vaccine are produced by inactivated pathogens.

Killed vaccines induce a predominantly humoral immune responses.

(1) Inactivated vaccines often require relatively large and repeated booster doses.

(2) Inactivated whole- organisms may remain associated with some active pathogens that may cause problems.

(3) Inactivated vaccines are less effective than attenuated vaccines.

2. Purified Antigen (Subunit) Vaccines:

Subunits vaccines are composed of specific antigenic macromolecules purified from pathogens and are usually administered with an adjuvant. In general, three forms of such vaccines are in current use:

(iii) recombinant antigen vaccines.

(i) Polysaccharide vaccines:

Such vaccines are produced from the hydrophilic polysaccharide capsule of some bacteria. Coating of such capsule with antibodies and/or complement greatly increases the ability of phagocytic cells to phagocytose such pathogens.

Although such poly­saccharide vaccines are inefficient inducers of B cell memory. These vaccines often provide long-lived protective immunity, probably because the polysaccharides are not degraded easily and they persist in lymphoid tissues and stimulate specific B cells for long periods.

High affinity antibody responses may be generated against polysaccharide antigens by coupling them to proteins to form conjugate vaccines.

The best known polysaccharide vaccines are used against Pneumococcus (causing pneumonia), and Haemophilus influenzae type b (Hib) (causing bacterial meningitis). Such vaccines have the limitation in their inabilities to activate TH cells. However, the Hib vaccine can activate the memory B cells to some degree in absence of a population of memory TH cells, thus establishing its efficacy as a fruitful vaccine.

(ii) Cytotoxin vaccines:

Many bacteria secrete exotoxins that cause many disease symptoms in the hosts. In preparation of cytotoxin vaccines, bacterial exotoxins are first purified and then chemically inactivated to form toxoid. Such toxoids when used as vaccines, these induce production of anti- toxoid antibodies, which can bind to exotoxins and neutralize their effects.

Diphtheria and tetanus toxoid vaccines are produced following such methods.

(iii) Recombinant antigen vaccines:

Genes encoding surface antigens of different pathogens like virus, bacteria and protozoa can be cloned and expressed successfully into suitable vectors. Such expressed antigens are then used for vaccine preparation.

The gene for the major surface antigen of hepatitis B virus (HBs Ag) was cloned in yeast cells. The resulting recombinant yeast cells then were grown in large fermenters HbsAg were found to accumulate within the yeast cells.

These yeast cells were collected and disrupted by high pressure and recombinant HBs Ag were collected and purified following biochemical techniques. Such recombinant HBs Ag is then used for development of the vaccines.

3. Live Recombinant Vector Vaccines:

A recent approach to vaccine deve­lopment is to introduce genes encoding microbial antigens into attenuated virus or bacteria (non-cytopathic vectors) and to infect individuals with such vectors. Thus, the vectors serve as sources of the antigen in the inoculated hosts.

One of the great advantages of such vaccines is that the vectors, like other live viruses or bacteria, induce the full comple­ment of immune responses, including strong CTL responses.

This technique has been used most commonly with vaccinia virus vectors, into which several dozen foreign genes can be inserted without impairing its capacity to infect host cells and replicate. Such genetically engineered vaccinia can express high levels of the inserted gene product, which can then serve as a potent immunogen in the inoculated host (the process for development of such vaccines are shown in Fig. 6.69).

Important virus vectors used for recombinant vaccine preparation include canary pox virus, attenuated poliovirus, attenuated strains of Salmonella (for cholera vaccine), BCG strain of Mycobacterium bovis and vaccinia virus (for smallpox vaccine).

A potential problem with viral vectors is that the viruses may infect various host cells, and even though they are not pathogenic, they can produce antigens that stimulate CTL responses, that kill the infected host cells.

The newest method of vaccination that has developed is the DNA vaccines that offer advantages over many of the existing vaccines. In this vaccination strategy, plasmid DNA encoding antigenic proteins are injected directly into the muscle of the recipient.

The DNA is taken up by muscle cells and the encoded protein antigen is expressed, leading to strong and long-lived humoral and cell-mediated immune responses to the antigen in the host.

The DNA appears either to integrate into the chromosomal DNA or to be maintained for long periods in an episomal form. The viral DNA is expressed not only by the muscle cells but it is likely that APCs, such as dendritic cells are also transfected by the plasmid and the cDNA is transcribed and translated into immunogenic protein that elicits specific response (See Fig. 6.70).

The unique feature of DNA vaccines is that they provide the only approach, other than live viruses, for eliciting strong CTL responses because the DNA-encoded proteins are synthesised in the cytosol of transfected cells.

Furthermore, bacterial plasmids are rich in un-methylated CpG nucleotides and are recognised by a Toll-like receptor (TLR9) on macrophages and other cells, thereby eliciting an innate immune response that enhances adaptive immunity. Therefore, plasmid DNA vaccines are effective even when they are administered without adjuvants.

The practical aspects of such vaccines are also very promising. Refrigeration is not required for the storage of the plasmid DNA, a feature that greatly reduces the cost and complexity of delivery. The same plasmid vector can be custom-tailored to make a variety of proteins, so that the same manufacturing techniques can be used for different DNA vaccines, each encoding an antigen from a different pathogen.

Such vaccines may be applied by coating microscopic gold beads with the plasmid DNA and then be delivered through the skin into the underlying muscle with an air-gun (gene gun), without requiring massive quantities of needles and syringes.

Results to date with DNA vaccines are very promising and at present there are human trials underway with several different DNA vaccines, including those for malaria, AIDS, influenza and herpes virus.

5. Synthetic peptide vaccines:

Synthetic peptides that represent immunodominant T- or B-cell epitopes are being evaluated as vaccines for several diseases. Since peptides are not as immunogenic as proteins, use of conjugates and adjuvants can assist in raising protective immunity to peptides. For the development of such vaccines, the vaccine designers select the peptides that compose immunodominant B-cell epitopes.

Such epitopes can be identified by determining the dominant antibody in the sera of individuals who are recovering from a disease and then testing various synthetic peptides for their ability to react with that antibody with a high affinity.

For example, two linear synthetic peptides representing potential B-cell epitopes of HBs Ag were tested for their binding affinity to pooled antisera from individuals who had recovered from hepatitis B.

In the practical experiment, it was found that a peptide with a cyclical repeats of amino acids 139-147 had a tenfold higher affinity than cyclical repeats of amino acids 124-137. Therefore, the cyclical peptide 139-147 was chosen as a potential one for the synthetic hepatitis B vaccine.

6. Multivalent subunit vaccines:

The above mentioned peptide vaccines are poorly immunogenic and they tend to induce mainly humoral immune responses. To overcome these limitations, a number of innovative techniques are being applied to develop multivalent vaccines that can present multiple copies of a given peptide or a mixture of peptides containing both immunodominant B-cell and T-cell epitopes.

In one approach, solid matrix-antibody antigen (SMAA) complexes are prepared by attaching monoclonal antibodies to particulate solid matrices and then saturating the anti­body with the designed antigen. The resulting complexes then can be used as vaccines.

In this way, by attaching different monoclonal antibodies to the solid matrix, it is possible to bind a mixture of peptides or proteins, compo­siting immunodominant epitopes for both T cells and B cells, to the solid matrix.

Such multivalent complexes can induce vigorous humoral and cell-mediated responses. Furthermore, the particulate nature of such vaccines increases the immunogenicity by facilitating phagocytosis by phagocytic cells.

In another approach, liposomes contai­ning protein antigens are prepared by mixing the proteins with a suspension of phospho­lipid under conditions that form vesicles bounded by a bilayer. The proteins are incorporated into the bilayer with the hydrophilic residues exposed.

Immunostumulating complexes (ISCOMs) are lipid carriers prepared by mixing protein or peptide antigens with detergent and a glycoside called Quil A. Following these approaches, peptides from various pathogens including influenza virus, hepatitis B virus, measles virus and HIV have been incorporated into liposomes and ISCOMs are currently being assessed as potential vaccines.

How Does Bacterial Invasion Lead to an Infection?


When an infection spreads through direct or indirect contact, it may lead to an infection. In order to differentiate between direct and indirect transmissions, the term contagious disease is used to specify a disease that is caused by direct contact. Infectious disease is a term that is generally used to specify diseases spread by other modes. Thus, sexually transmitted diseases are examples of infections spread by direct contact. Infections spread by contact with infected objects like pencils, glasses, towels, toys, etc. are termed as indirect infections like in the case of diphtheria.


Most of the respiratory infections are spread through inhalation of the infectious bacteria. These bacteria tend to be present in air in form of aerosols. They are released in the environment by sneezing, coughing, talking, spitting, etc. Most of the time these respiratory droplets dry off. However, some bacteria are resistant to drying and can remain suspended in air for long period. Thus, when a healthy person inhales these droplets, it may lead to respiratory infection.


Gastrointestinal infections are usually caused by ingestion of the pathogens or their toxins. Thus, giving rise to different illnesses like waterborne, food-borne and hand-borne illnesses. These pathogens enter the gastrointestinal tract through the mouth. Examples of diseases caused through ingestion include cholera, dysentery and food poisoning.


When bacteria is inoculated into the subcutaneous body tissue, it may lead to infection. For example, a deep wound may give a chance to Clostridium tetani a chance to cause a tetanus infection. Similarly, gangrene causing bacteria may also cause cellular death and tissue decay.


Pathogens that are able to cross the placental barrier and infect the fetus in the uterus are called congenital infections. These infections can lead to congenital disorders in the baby.

Bacterium causing rabbit fever remains virulent for months in cold water

Northern Arizona University professor David Wagner, director of the Pathogen and Microbiome Institute's (PMI) Biodefense and Disease Ecology Center, led a study to better understand the life cycle and behavior of F. tularensis, funded through a $2.25 million grant from the U.S. Defense Threat Reduction Agency (DTRA). Credit: Northern Arizona University

Although it is not spread through human contact, Francisella tularensis is one of the most infectious pathogenic bacteria known to science—so virulent, in fact, that it is considered a serious potential bioterrorist threat. It is thought that humans can contract respiratory tularemia, or rabbit fever—a rare and deadly disease—by inhaling as few as 10 airborne organisms.

Northern Arizona University professor David Wagner, director of the Pathogen and Microbiome Institute's (PMI) Biodefense and Disease Ecology Center, began a three-year project in 2018 to better understand the life cycle and behavior of F. tularensis, funded through a $2.25 million grant from the U.S. Defense Threat Reduction Agency (DTRA).

One of the most puzzling behaviors of the pathogen is its ability to remain dormant, possibly in what is called a "viable but nonculturable" state—which means the bacteria is alive, but cannot be grown in the laboratory. That makes it much more difficult to study, because scientists can typically only study bacteria that can be cultured. Wagner's goal was to study the bacterium so as to determine the environmental and genetic factors that contribute to the pathogen's ability to apparently remain dormant for months at a time—a phenomenon that has remained mostly a mystery despite more than 100 years of research.

Now, Wagner and his collaborators have published their findings, "Long-Term Survival of Virulent Tularemia Pathogens outside a Host in Conditions That Mimic Natural Aquatic Environments," in the journal Applied and Environmental Microbiology. In the paper, the team shows how they were able to prove, by replicating environmental conditions in the lab, including low temperatures and low-nutrient water, that the bacterium can persist for months in cold water without any nutrients and remain fully virulent. Their results provide a plausible explanation for how it can overwinter in the environment outside of a host.

"We are making some very interesting discoveries in this project," Wagner said. "The main finding is that Francisella tularensis can persist in a dormant state for more than six months in cold water without any nutrients. This means it has the ability to persist in the environment outside of a mammalian host or arthropod vector. This was unexpected because many other bacteria that persist like that long-term in the environment form spores when they are outside of a host, such as Bacillus anthracis, the bacterium that causes anthrax forms spores, but F. tularensis doesn't do that. Others, like Yersinia pestis—the bacterium that causes plague—are always either in a mammalian host or a flea vector. F. tularensis has the ability to persist long-term in the environment long-term outside of a host without forming spores while remaining fully virulent."

"These study results have completely changed our perspective on the ecology of this bacterium. We now understand that mammals are likely just a small (but still important) aspect of its survival strategy. We now think that it spends most of its time in the environment outside of a host and only periodically causes disease in mammals. But those disease events in mammals are still very important as they serve to amplify the amount of F. tularensis that is deposited back in the environment."

Working with co-principal investigator Jason Sahl, associate professor and assistant director of PMI, and with PMI senior research scientists Dawn Birdsell and Joe Busch, Wagner conducted the study along with colleagues at two of the team's long-term collaborating institutions in Sweden: The Swedish Defence Research Agency and Umeå University.

Along with their Swedish collaborators, Wagner and his team are known worldwide for their work developing the phylogeny, or global family tree, of F. tularensis and its phylogeography—mapping where different groups of the species are found throughout the world and understanding the species' genetic diversity.

"As we continue with the DTRA research grant, we are now investigating the genes and proteins that regulate the ability of F. tularensis to persist in the environment outside of mammals and hosts. This work involves a number of current and recently graduated undergraduate students at NAU: Former student Kathleen Soria, current students Natalie Hart and Rebecca Ballard, and current student and Flinn Scholar Kailee Savage.

Marine pathogenic bacterium forms specialized cells for dissemination

Its single polar flagellum enables Vibrio parahaemolyticus to swim and spread in the ocean. The image shows a transmission electron micrograph of a polarly flagellated cell in the process of cell division, as visualized by the constriction in the middle of the cell. Credit: MPI f. terrestrial Microbiology/ Ringaard

Vibrio parahaemolyticus can be found in the tidal zones in estuarine areas. The marine bacterium causes acute gastroenteritis in humans and is the leading cause for seafood borne illnesses in the world. Researchers from the Max Planck Institute for terrestrial Microbiology in Marburg, Germany, have identified specialized "adventurer" cells that ensure the bacterium's dissemination and prevalence. Their new findings are an important basis for the future management of the disease.

In Central and Northern Europe, Vibrio infections are among the "emerging diseases" whose incidence has recently increased or is likely to increase in the near future. Some reasons for this are global trade and the higher water temperatures caused by global climate change. Mussels, oysters and crabs that are found in our supermarkets from tropical regions are possibly contaminated all year round and to a high percentage. They can cause an infection if eaten raw or are insufficiently cooked.

Vibrio parahaemolyticus forms colonies in the tidal zone of estuarine areas, and its complex life cycle is triggered by the respective conditions of this habitat. But how does the species adapt to environmental changes, and how can it colonize new habitats? "In order to develop any measures against the spread of Vibrio parahaemolyticus and related bacteria, we must first understand the structure and distribution strategy of the bacterial colonies," explains Simon Ringgaard of the Max Planck Institute for Terrestrial Microbiology in Marburg. In their laboratory, he and his team simulate the conditions of the tidal zone and thus investigate the bacterial life cycle and mechanisms of movement.

Development of swarm colonies and dissemination of adventurer cells in the environment. 0) Planktonic cell attaching to a solid surface Under swarm inducing conditions, cells can initiate the development of a swarm colony which consists of four stages 1) Stage I of swarm colony development: colony growth 2) Stage II of development: differentiation initiation and swarm-flare formation 3) Stage III of development: swarm-front expansion 4) Stage IV of development: swarm colony maturation and final architecture formation. 5) Once a mature swarm colony is flooded, a morphologically short and specific cell type is released into the liquid – the adventurer cells. Adventurer cells are highly swimming proficient and can swim towards chitin (a component of seafood) and attach to it. Credit: MPI f. terrestrial Microbiology/ Duarte deFreitas

Swimmer and swarm cells

As many other bacteria, Vibrio parahaemolyticus forms special cell types when environmental conditions require it. While short swimmer cells with a single polar flagellum can move quickly in a liquid environment, the longer swarm cells reside within bacterial populations that are attached to solid surfaces. Swarmer cells are specialized for movement over surfaces and can rapidly colonize new surface areas.

The Vibrio bacterial swarm colonies show a distinct stratification: while the middle of the colony consists of rather shorter cells, the longer swarm cells are found in the outer areas of the colony. As the Max Planck researchers were able to show, if the swarm colony is flooded with water, as in the natural habitat during the tidal rhythms, cells are released from the colony into the liquid surroundings. Surprisingly, however, these released cells are neither the long, swarmer cells nor the very short cells found in the middle, but a completely unexpected and new cell type of medium length. These "adventurer cells" are optimized for living in water and possess particularly good swimming properties.

The research team showed that once released, the adventurer cells were highly capable of spreading in their new liquid environments and importantly they were able to "smell" and move towards potential nutrient sources such as chitin—an essential component of marine animals to which Vibrio parahaemolyticus attaches. Thus, the release of adventurer cells into the water has the potential to help spread the bacterium in the environment and bring Vibrio parahaemolyticus to new shores, like to the surface of seafood. And thus into our food chains, consequentially likely enhancing the risk of human infections.

The Marburg researchers investigated the life cycle as a function of environmental conditions and time, both morphologically and on the molecular genetic level. Here they found characteristic expression patterns that could also be used for the future detection of the bacterium. But perhaps this involves even something much more far-reaching, says Simon Ringgaard. "Our experiments show that the colony always has a sub-population of adventurer cells that are ready to be released immediately upon flooding. Adventurer cells would thus be of central importance for the worldwide epidemiology of the disease—and thus also for measures to contain it, for example in industrial aquaculture."

Cryptococcus neoformans Chitin Synthase 3 Plays a Critical Role in Dampening Host Inflammatory Responses

Cryptococcus neoformans infections are significant causes of morbidity and mortality among AIDS patients and the third most common invasive fungal infection in organ transplant recipients. One of the main interfaces between the fungus and the host is the fungal cell wall. The cryptococcal cell wall is unusual among human-pathogenic fungi in that the chitin is predominantly deacetylated to chitosan. Chitosan-deficient strains of C. neoformans were found to be avirulent and rapidly cleared from the murine lung. Moreover, infection with a chitosan-deficient C. neoformans strain lacking three chitin deacetylases (cda1Δcda2Δcda3Δ) was found to confer protective immunity to a subsequent challenge with a virulent wild-type counterpart. In addition to the chitin deacetylases, it was previously shown that chitin synthase 3 (Chs3) is also essential for chitin deacetylase-mediated formation of chitosan. Mice inoculated with the chs3Δ strain at a dose previously shown to induce protection with the cda1Δcda2Δcda3Δ strain die within 36 h after installation of the organism. Mortality was not dependent on viable fungi, as mice inoculated with a heat-killed preparation of the chs3Δ strain died at the same rate as mice inoculated with a live chs3Δ strain, suggesting that the rapid onset of death was host mediated, likely caused by an overexuberant immune response. Histology, cytokine profiling, and flow cytometry indicate a massive neutrophil influx in the mice inoculated with the chs3Δ strain. Mice depleted of neutrophils survived chs3Δ inoculation, indicating that death was neutrophil mediated. Altogether, these studies lead us to conclude that Chs3, along with chitosan, plays critical roles in dampening cryptococcus-induced host inflammatory responses.IMPORTANCE Cryptococcus neoformans is the most common disseminated fungal pathogen in AIDS patients, resulting in ∼200,000 deaths each year. There is a pressing need for new treatments for this infection, as current antifungal therapy is hampered by toxicity and/or the inability of the host's immune system to aid in resolution of the disease. An ideal target for new therapies is the fungal cell wall. The cryptococcal cell wall is different from the cell walls of many other pathogenic fungi in that it contains chitosan. Strains that have decreased chitosan are less pathogenic and strains that are deficient in chitosan are avirulent and can induce protective responses. In this study, we investigated the host responses to a chs3Δ strain, a chitosan-deficient strain, and found that mice inoculated with the chs3Δ strain all died within 36 h and that death was associated with an aberrant hyperinflammatory immune response driven by neutrophils, indicating that chitosan is critical in modulating the immune response to Cryptococcus.

Keywords: chitin chitin synthase chitosan inflammation neutrophils.

Copyright © 2020 Hole et al.


Deletion and complementation of C.…

Deletion and complementation of C. neoformans chitin synthase 3 (Chs3). (A) For morphological…

Inoculation with the chs3Δ strain…

Inoculation with the chs3Δ strain induces rapid mouse mortality. C57BL/6 mice were infected…

Mortality is not dependent on…

Mortality is not dependent on the viability of the fungi or mouse background.…

A massive inflammatory response is…

A massive inflammatory response is triggered by chs3 Δ. C57BL/6 mice were inoculated…

A significant increase in neutrophil…

A significant increase in neutrophil recruitment in chs3Δ -inoculated mice. C57BL/6 mice were…

Depletion of neutrophils protects chs3Δ…

Depletion of neutrophils protects chs3Δ -inoculated mice. (A to C) C57BL/6 (A), BALB/c…