MicrobiologyBytes

Salmonella enterica serotype Typhimurium (S. Typhimurium) is an important food-borne pathogen that in humans causes a self-limited gastroenteritis, characterized by fever, acute intestinal inflammation, diarrhea, and the presence of neutrophils in stool samples. In addition, S. Typhimurium is a model organism for studying bacterial genetics and microbial pathogenesis. As the frontier in bacterial pathogenesis research is moving towards understanding the complexity of host-pathogen interaction at the tissue level, studies on the pathogenesis of S. Typhimurium gastroenteritis using animal models have helped establish important new concepts that exert a strong influence on the research field. Recent studies on S. Typhimurium pathogenesis reveal how tissue-specific host factors and the presence of other bacterial species shape the outcome of host-pathogen interaction in the intestinal lumen. This review discusses these new paradigms for the interplay between the pathogen, the host and its resident microbial community.
Salmonella, the host and its microbiota. Curr Opin Microbiol. Oct 24 2011
The intestine is host to a diverse bacterial community whose structure, at the phylum level, is maintained through unknown mechanisms. Acute inflammation triggered by enteric pathogens, such as Salmonella enterica serotype Typhimurium (S. Typhimurium), is accompanied by changes in the bacterial community structure marked by an outgrowth of the pathogen. Recent studies show that S. Typhimurium can harness benefit from the host response to edge out the beneficial bacterial species that dominate in the healthy gut. The elucidation of how S. Typhimurium alters the bacterial community structure during gastroenteritis is beginning to provide insights into mechanisms that dictate the balance between the host and its microbiota.

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When it comes to nasty pathogens, Marburg virus is among the nastiest. Cousin to Ebola virus, Marburg causes fever, rash, delirium, and severe hemorrhaging, often ending in organ failure and death. It is rare in the wild, but was a central focus of weaponization by the Soviet Union, and remains a concern for terrorism experts who fear its lethal potential and resistance to treatment.

One reason that treatments have proved so elusive is because the virus is so hard to work with – hazmat suits, self-contained breathing gear, and electronically secured airlocks are all required for even the simplest of studies with live virus. But another reason is that the virion (the virus particle) is heterogeneous in shape, and that heterogeneity has confounded standard imaging techniques (X-ray crystallography, cryo-electron microscopy), which require purified, identical particles to obtain their highest resolution. Researchers have now got around that problem by using a sophisticated combination of imaging techniques that provide the first clear three-dimensional picture of the intact Marburg virion structure.

Marburg Virus Structure Revealed in Detail. (2011) PLoS Biol 9(11): e1001198. doi:10.1371/journal.pbio.1001198
and:
Cryo-Electron Tomography of Marburg Virus Particles and Their Morphogenesis within Infected Cells. (2011) PLoS Biol 9(11): e1001196. doi:10.1371/journal.pbio.1001196
Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible “Velcro-like” manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.

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On Friday afternoon I gave first year students a leture about microbiology (yes, all of it :-) Along the way, I touched on the idea of the post-antibiotic era and posed a question as to whether nanotechnology might be able to rescue the failing antimicrobials pipeline. The truth is, I don’t really know that much about nanotechology, but The Guardian has a rather good introductory guide this weekend, Nanotechnology World, sponsored by NanoChannels:

Nanotechnology World

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The introduction of rRNA-targeted fluorescence in situ hybridization (FISH) using oligonucleotide probes for the cultivation-independent identification of microbes more than 20 years ago marked the beginning of a new era for environmental and medical microbiology. When integrated into the so-called full-cycle rRNA approach, FISH enables microbiologists to decipher complete structures of microbial communities in a quantitative manner. Furthermore, this phylogenetic staining technique in its basic format is easy to apply and once probes have been designed and evaluated, the detection of their target organisms in environmental or medical samples is straightforward and can be completed in a few hours. In its original format, fluorescent monolabeled oligonucleotide probes are used for FISH, but as the signal intensity of this technique is insufficient for cells with low ribosome contents, FISH detection efficiencies in oligotrophic environments are generally rather low. For such systems, catalyzed reporter deposition (CARD)-FISH, which exploits horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification is the method of choice to capture most microbial community members.

rRNA-targeting FISH techniques are continuously developed further and major improvements regarding increased cell permeability, accessibility of probe target sites, probe specificity, signal intensity, and so on have been achieved. A second rapidly evolving FISH-related research area is the combination of rRNA-FISH with other techniques, which provide additional information on (i) the presence of specific genes or mRNA molecules of the target cell, (ii) its specific metabolic activity or (iii) important environmental parameters such as the concentration of chemical compounds in the vicinity of the detected cell. For this purpose rRNA-FISH or CARD-FISH have been combined with various other FISH techniques and staining procedures as well as with microautoradiography, microelectrode measurements, Raman microspectroscopy, and NanoSIMS. This review provides an overview on the most recent developments in the FISH field.

New trends in fluorescence in situ hybridization for identification and functional analyses of microbes. Curr Opin Biotechnol. Nov 11 2011
Fluorescence in situ hybridization (FISH) has become an indispensable tool for rapid and direct single-cell identification of microbes by detecting signature regions in their rRNA molecules. Recent advances in this field include new web-based tools for assisting probe design and optimization of experimental conditions, easy-to-implement signal amplification strategies, innovative multiplexing approaches, and the combination of FISH with transmission electron microscopy or extracellular staining techniques. Further emerging developments focus on sorting FISH-identified cells for subsequent single-cell genomics and on the direct detection of specific genes within single microbial cells by advanced FISH techniques employing various strategies for massive signal amplification.

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Epstein-Barr virus (EBV) and Kaposi’s Sarcoma Associated Herpesvirus (KSHV) are DNA tumor viruses that provide risk factors for Burkitt’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma, Kaposi’s Sarcoma and post-transplant lymphoproliferative disease. EBV infection has also been associated with multiple sclerosis. Healthy carriers consistently shed virus in saliva that infects naïve individuals despite being exposed to virus-specific antibody. This lack of neutralization contrasts completely with non-persistent mucosal infections such as that of poliovirus, and implies that gammaherpesviruses have evolved specific antibody evasion mechanisms.

Our understanding of EBV and KSHV is limited by their narrow species tropisms. Related animal viruses are therefore an important source of information. Two of the best established experimental models are provided by Murid herpesvirus 4 (MuHV-4) and Bovine herpesvirus 4 (BoHV-4). The homologs of gp350 are gp150 in MuHV-4 and gp180 in BoHV-4 are diverse in sequence but seem to be related in function, being involved in both binding to a cellular receptor and in blocking the infection of cells that do not express this receptor. So a non-essential glycoprotein hides some epitopes on cell-free virions from neutralization.

Antibody Evasion by a Gammaherpesvirus O-Glycan Shield. (2011) PLoS Pathog 7(11): e1002387. doi:10.1371/journal.ppat.1002387
All gammaherpesviruses encode a major glycoprotein homologous to the Epstein-Barr virus gp350. These glycoproteins are often involved in cell binding, and some provide neutralization targets. However, the capacity of gammaherpesviruses for long-term transmission from immune hosts implies that in vivo neutralization is incomplete. In this study, we used Bovine Herpesvirus 4 (BoHV-4) to determine how its gp350 homolog – gp180 – contributes to virus replication and neutralization. A lack of gp180 had no impact on the establishment and maintenance of BoHV-4 latency, but markedly sensitized virions to neutralization by immune sera. Antibody had greater access to gB, gH and gL on gp180-deficient virions, including neutralization epitopes. Gp180 appears to be highly O-glycosylated, and removing O-linked glycans from virions also sensitized them to neutralization. It therefore appeared that gp180 provides part of a glycan shield for otherwise vulnerable viral epitopes. Interestingly, this O-glycan shield could be exploited for neutralization by lectins and carbohydrate-specific antibody. The conservation of O-glycosylation sites in all gp350 homologs suggests that this is a general evasion mechanism that may also provide a therapeutic target.

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Gathering and sharing of information is extremely important in human society. Especially in times of war, the difference between victory and defeat can depend on the ability to obtain, encrypt, and share information, and sophisticated systems have been developed for exactly this purpose. Similarly, in their constant battles with competitors and the host immune system, (opportunistic) microbial pathogens have developed sophisticated cell–cell communication systems termed quorum sensing (QS) that allow exchange of critical information. In return, competing microbes, as well as the host immune system, have developed means to intercept and decode these messages. The information obtained by this molecular espionage is used for their benefit, either to win the war (microbe against microbe), or to prepare for an upcoming battle (microbe against immune system).

QS is a system that enables microbes to monitor population cell density through the production, secretion, and sensing of small diffusible molecules. When such molecules reach a threshold concentration, microbial cells in the vicinity detect the signal and coordinately respond by modifying their gene expression; often these genes are associated with virulence and pathogenesis. Several different types of QS molecules have been described for a wide variety of microbial species.

To illustrate the clinical importance of this microbial spy game, this short review focuses on the biological activity of a single bacterial QS molecule on surrounding microbes and the host immune system and its diverse “meaning” to different receivers. Infections related to burn wounds, cystic fibrosis, and periodontal diseases consist most commonly of the bacteria Pseudomonas aeruginosa and Staphylococcus aureus and the fungus Candida albicans, and represent niches with an active host response. This short review provides five facts about how the P. aeruginosa QS molecule plays a pivotal role in this triangle of interspecies interactions and how microbial behavior elicited by this small signalling molecule has consequences for the host response.

Microbial Spy Games and Host Response: Roles of a Pseudomonas aeruginosa Small Molecule in Communication with Other Species. (2011) PLoS Pathog 7(11): e1002312. doi:10.1371/journal.ppat.1002312

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Carbapenems were the last β-lactams retaining near-universal anti-Gram-negative activity, but carbapenemases are spreading, conferring resistance. New Delhi metallo-β-lactamase (NDM) enzymes are the latest carbapenemases to be recognized and since 2008 have been reported worldwide, mostly in bacteria from patients epidemiologically linked to the Indian subcontinent, where they occur widely in hospital and community infections, and also in contaminated urban water. The main type is NDM-1, but minor variants occur. NDM enzymes are present largely in Enterobacteriaceae, but also in non-fermenters and Vibrionaceae. Dissemination predominantly involves transfer of the bla(NDM-1) gene among promiscuous plasmids and clonal outbreaks. Bacteria with NDM-1 are typically resistant to nearly all antibiotics, and reliable detection and surveillance are crucial.

E. coli is one of the most prevalent human pathogens, and the window of opportunity to control it from becoming widely resistant is rapidly closing. No vaccine is likely to become available and one that affects commensal gut strains would probably be undesirable, even though these might act as vectors of potent resistance, including NDM-1. Therefore, everything must be done now to prevent infections due to bacteria with NDM-1, otherwise infections as common as pyelonephritis might soon become life-threatening owing to the lack of effective treatment.

The emerging NDM carbapenemases. Trends Microbiol. Nov 9 2011

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Cell-cell interactions are common to all living systems. Bacteria are no exception, and numerous mechanisms that use secreted products as signaling molecules are known. Among these, the so-called “quorum sensing” systems are perhaps the best studied. In quorum sensing, all bacterial cells within a population produce secreted molecules. Only when population densities are high is there a response to these compounds, thus allowing the bacteria to coordinate their behavior. However, it is clear that there is much more to bacterial cell–cell interactions than simply counting numbers and coordinating behavior. Secreted molecules also play key roles in microbial development so that different cell fates can arise and coexist within a single-species population. In addition, in settings where multiple species coexist, their interactions often are mediated through extracellular compounds. Development in one microbe can be influenced by small molecules secreted by other species.

Almost all quorum sensing studies have been performed under laboratory conditions – far removed from how bacteria actually live. What happens in the “wild” when rival bacterial gangs contest the same territory?

Interspecies interactions that result in Bacillus subtilis forming biofilms are mediated mainly by members of its own genus. PNAS USA November 10 2011. doi: 10.1073/pnas.11036301
Many different systems of bacterial interactions have been described. However, relatively few studies have explored how interactions between different microorganisms might influence bacterial development. To explore such interspecies interactions, we focused on Bacillus subtilis, which characteristically develops into matrix-producing cannibals before entering sporulation. We investigated whether organisms from the natural environment of B. subtilis – the soil – were able to alter the development of B. subtilis. To test this possibility, we developed a coculture microcolony screen in which we used fluorescent reporters to identify soil bacteria able to induce matrix production in B. subtilis. Most of the bacteria that influence matrix production in B. subtilis are members of the genus Bacillus, suggesting that such interactions may be predominantly with close relatives. The interactions we observed were mediated via two different mechanisms. One resulted in increased expression of matrix genes via the activation of a sensor histidine kinase, KinD. The second was kinase independent and conceivably functions by altering the relative subpopulations of B. subtilis cell types by preferentially killing noncannibals. These two mechanisms were grouped according to the inducing strain’s relatedness to B. subtilis. Our results suggest that bacteria preferentially alter their development in response to secreted molecules from closely related bacteria and do so using mechanisms that depend on the phylogenetic relatedness of the interacting bacteria.

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Cowpox virus (CPXV) is one of the earliest described members of the genus Orthopoxvirus (OPV). Historically, researchers referred to the ailment known as cowpox and even suggested that it could provide immunity against smallpox. It was Edward Jenner’s publications in 1798 and 1799 which provided the first scientific description of vaccination by detailing the efficacy of CPXV “scarification” in inducing protective immunity against challenge with variola (smallpox) virus (VARV). The common name “cowpox virus” refers to the association with pustular lesions on the teats of cows and historic zoonotic transmission of this disease to humans (milkers) through contact with infected cows. Human infections are generally mild and self-limiting with localized skin lesions healing after 3–4 weeks, however, systemic involvement and fatal outcome have been reported in immunocompromised individuals.

New analysis shows that the smallpox vaccine is known to have originated in the United Kingdom, however the vaccine strains were most closely allied to CPXV isolates from Russia and from Finland. The most likely scenario is that most of the commercially produced smallpox vaccines were not made from the original Jenner strain, but instead from isolates found in other regions of Europe.

Chasing Jenner’s Vaccine: Revisiting Cowpox Virus Classification. (2011) PLoS ONE 6(8): e23086. doi:10.1371/journal.pone.0023086
Cowpox virus (CPXV) is described as the source of the first vaccine used to prevent the onset and spread of an infectious disease. It is one of the earliest described members of the genus Orthopoxvirus, which includes the viruses that cause smallpox and monkeypox in humans. Both the historic and current literature describe “cowpox” as a disease with a single etiologic agent. Genotypic data presented herein indicate that CPXV is not a single species, but a composite of several (up to 5) species that can infect cows, humans, and other animals. The practice of naming agents after the host in which the resultant disease manifests obfuscates the true taxonomic relationships of “cowpox” isolates. These data support the elevation of as many as four new species within the traditional “cowpox” group and suggest that both wild and modern vaccine strains of Vaccinia virus are most closely related to CPXV of continental Europe rather than the United Kingdom, the homeland of the vaccine.

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