MicrobiologyBytes

The production of new HIV-1 particles is initiated at the plasma membrane where the virus polyprotein Gag assembles into a budding site, and proceeds through release of an immature virion which is subsequently transformed to the infectious virion by proteolytic cleavage of Gag. Recently HIV-1 budding sites have been studied by cryo electron tomography. This technique allows three-dimensional structure determination of single objects at macromolecular resolution, and is uniquely suited to study variable structures such as HIV-1 particles and budding sites.

Using cryo electron tomography, researchers obtained three-dimensional images with unprecedented detail of the formation of HIV-1 particles. By analyzing these images they showed that the organization of released immature HIV-1 is determined at its intracellular assembly without major subsequent rearrangements. They were able to identify a lattice structure of the viral protein Gag present in budding sites that seem to lack the viral genome and thus cannot be precursors of infectious viruses. Some HIV-1 infected T-cells preferentially carry these budding sites, suggesting that they have lost a crucial control of the proteolytic maturation of the virus.

Cryo Electron Tomography of Native HIV-1 Budding Sites. (2010) PLoS Pathog 6(11): e1001173. doi:10.1371/journal.ppat.1001173
The structure of immature and mature HIV-1 particles has been analyzed in detail by cryo electron microscopy, while no such studies have been reported for cellular HIV-1 budding sites. Here, we established a system for studying HIV-1 virus-like particle assembly and release by cryo electron tomography of intact human cells. The lattice of the structural Gag protein in budding sites was indistinguishable from that of the released immature virion, suggesting that its organization is determined at the assembly site without major subsequent rearrangements. Besides the immature lattice, a previously not described Gag lattice was detected in some budding sites and released particles; this lattice was found at high frequencies in a subset of infected T-cells. It displays the same hexagonal symmetry and spacing in the MA-CA layer as the immature lattice, but lacks density corresponding to NC-RNA-p6. Buds and released particles carrying this lattice consistently lacked the viral ribonucleoprotein complex, suggesting that they correspond to aberrant products due to premature proteolytic activation. We hypothesize that cellular and/or viral factors normally control the onset of proteolytic maturation during assembly and release, and that this control has been lost in a subset of infected T-cells leading to formation of aberrant particles.

Related:

• HIV-1 Assembly and Release
• Studying the structure of HIV
• Giant microscope meets giant virus

Mycobacterium tuberculosis (Mtb) has the ability to lie dormant in the human body for decades, only progressing to active disease in 5–10% of immunocompetent individuals. The organism is transmitted through aerosols, and enters the pulmonary system through inhalation. Within the lung, the bacillus can take up residence inside an alveolar macrophage triggering the aggregation of immune cells and the formation of a granuloma. During the course of infection, granulomas play a dual role – serving as a niche for the invading bacteria, whilst, protecting the host from active disease. The population of granulomas within the infected host consists of both primary and post-primary lesions. Primary granulomas containing the inhaled founder strain are morphologically different from post-primary granulomas that have developed through disseminated infection. This results in a heterogeneous population of bacilli that are unique to the in vivo experience

Portrait of a Pathogen: The Mycobacterium tuberculosis Proteome In Vivo. (2010) PLoS ONE 5(11): e13938. doi:10.1371/journal.pone.0013938
Background: Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a facultative intracellular pathogen that can persist within the host. The bacteria are thought to be in a state of reduced replication and metabolism as part of the chronic lung infection. Many in vitro studies have dissected the hypothesized environment within the infected lung, defining the bacterial response to pH, starvation and hypoxia. While these experiments have afforded great insight, the picture remains incomplete. The only way to study the combined effects of these environmental factors and the mycobacterial response is to study the bacterial response in vivo.
Methodology/Principal Findings: We used the guinea pig model of tuberculosis to examine the bacterial proteome during the early and chronic stages of disease. Lungs were harvested thirty and ninety days after aerosol challenge with Mtb, and analyzed by liquid chromatography-mass spectrometry. To date, in vivo proteomics of the tubercle bacillus has not been described and this work has generated the first large-scale shotgun proteomic data set, comprising over 500 unique protein identifications. Cell wall and cell wall processes, and intermediary metabolism and respiration were the two major functional classes of proteins represented in the infected lung. These classes of proteins displayed the greatest heterogeneity indicating important biological processes for establishment of a productive bacterial infection and its persistence. Proteins necessary for adaptation throughout infection, such as nitrate/nitrite reduction were found at both time points. The PE-PPE protein class, while not well characterized, represented the third most abundant category and showed the most consistent expression during the infection.
Conclusions/Significance: Cumulatively, the results of this work may provide the basis for rational drug design – identifying numerous Mtb proteins, from essential kinases to products involved in metal regulation and cell wall remodeling, all present throughout the course of infection.

Related:

• Systems biology approaches to tuberculosis
• Do mycobacteria produce endospores?
• Why is TB more common in men than in women?

There are estimated to be on the order of 10¹⁰ phage per liter of sea water and roughly 10²⁴ phage infections per second. Despite the frequency of phage infection and its ecological importance, relatively little is known about the molecular mechanics of the infection process. Questions such as what provides the driving force for genome exit from the capsid, what signals the conduit to open to allow exit and how the nucleic acid enters the host cell during infection remain unanswered. However, advances in electron microscopy and image analysis are allowing us to capture a glimpse of this remarkable process. The T7-like podovirus P-SSP7 infects Prochlorococcus marinus, the most abundant photosynthetic microorganism. A recent cryo-electron microscopy study provides insight into the molecular details of the P-SSP7 infection process, and given the similarity between P-SSP7 and other podoviruses is likely to provide a paradigm for understanding the process of phage infection.

Mind the Gap: How Some Viruses Infect Their Hosts. (2010) Viruses 2(11): 2536-2540; doi:10.3390/v2112536
Cryo-electron microscopy (Cryo-EM) and cryo-electron tomography (Cryo-ET) provide structural insights into complex biological processes. The podoviridae are dsDNA containing phage with short, non-contractile tails which nevertheless translocate their DNA into the cytoplasm of their host cells. Liu et al. [1] used a combination of cryo-EM and cryo-ET to study the structural changes accompanying infection of P. marinus by the phage P-SSP7 and thereby provide unique molecular insight into the process by which the DNA transits from phage to host during infection.

Related:

• Modelling bacteriophage
• Holins and the phage infection cycle
• Bacteriophage resistance

Bacterial pathogens exploit a huge range of niches within their hosts. Many pathogens can invade non-phagocytic cells and survive within a membrane-bound compartment. However, only a small number of bacteria, including Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis and Rickettsia spp., can gain access to and proliferate within the host cell cytosol. This review discusses the mechanisms by which these cytosolic pathogens escape into the cytosol, obtain nutrients to replicate and subvert host immune responses.

Residing within the cytosol may provide protection for bacteria against certain aspects of host defence, such as recognition by circulating antibodies, the complement system and the microbicidal environment of the phagolysosome. However, it is becoming increasingly clear that pathogens do not entirely escape the human immune system while in the cytosol. A series of intracellular receptors detect microbial pathogen-associated molecular patterns, and have evolved to combat both viral and bacterial pathogens. Although cytosolic pathogens have recently been shown to manipulate autophagic responses, they probably also interfere with many other aspects of host cell signalling to promote their survival within the cell. Therefore, the cytosol provides bacteria with only a limited subset of nutrients and presents them with the formidable challenge of evading an organized system of pathogen recognition mechanisms and responses. Future work will unveil further strategies that bacteria have successfully evolved to survive in and adapt to this specific host niche.

Life on the inside: the intracellular lifestyle of cytosolic bacteria. (2009) Nature Reviews Microbiology 7: 333-340 doi:10.1038/nrmicro2112

Related:

• Intracellular bacterial replication at the single cell level
• Video/Animations of Intracellular Pathogens
• Listeria monocytogenes replication in macrophage vacuoles

Chlamydia is the most prevalent bacterial sexually transmitted infection (STI) in the western world. In Ireland, the number of chlamydia notifications increased from 245 in 1995 to 6290 in 2008. While this may reflect a real rise in the burden of chlamydia infection, it also reflects an increase in provider awareness in chlamydia testing, test performance and the introduction of laboratory notification. The real burden (numbers of infection) is likely to be higher than reported as chlamydia is asymptomatic in approximately 70% of women and 50% of men and thus may remain undiagnosed. Prevalence studies in young Irish people (including students) have shown similar rates of infection to the UK and else where in Europe.

In view of the asymptomatic nature of chlamydia, especially in women, there is recognition that it is important to screen sexually active women aged less than 25 years. Two approaches are proposed: systematic, where all eligible young persons are systematically invited for screening, which requires the availability of a unique identifier for each individual to ensure all eligible persons are invited and not invited again except where indicated; and opportunistic, where eligible young persons that are visiting/utilising clinical and non-clinical settings are invited to take a test.

These issues highlight the need for additional strategies for screening that are free at the point of testing, easy to access, private and available in a variety of settings. Screening strategies need to be ‘youth friendly’ and available outside of traditional medical facilities.

Pee-in-a-Pot: acceptability and uptake of on-site chlamydia screening in a student population in the Republic of Ireland. (2010) BMC Infectious Diseases 10: 325 doi:10.1186/1471-2334-10-325
Background: The aim of the study was to explore the acceptability and uptake of on-campus screening using a youth friendly approach in two Third Level higher education institutions (HEIs). This study is part of wider research exploring the optimal setting for chlamydia screening in Ireland.
Methods: Male and female students were given the opportunity to take a free anonymous test for chlamydia during a one week programme of “pee-in-a-pot” days at two HEI campuses in the West of Ireland. The study was set up after extensive consultation with the two HEIs and advertised on the two campuses using a variety of media in the two weeks preceding the screening days. Screening involved the provision and distribution of testing packs at communal areas and in toilet facilities. In Ireland, chlamydia notifications are highest amongst 20-29 year olds and hence the screening criterion was aimed at 18-29 year olds. Urine samples were tested using a nucleic acid amplification test (NAAT). Following the screening days, qualitative in-depth interviews were conducted with participants about their experiences of the event.
Results: Out of 1,249 test kits distributed in two HEIs, 592 specimens were collected giving a return rate of 47.5%. Tests excluded (54) were due to labelling errors or ineligibility of participants’ age. Two thirds of those tested were females and the mean age was 21 years. Overall, 3.9% (21/538) of participants tested positive, 5% (17/336) among females and 2% (4/191) among males. Participant interviews identified factors which enhanced student participation such as anonymity, convenience, accessibility of testing, and the informal and non-medical approach to testing.
Conclusions: Screening for chlamydia using on-campus “pee-in-a-pot” days is an acceptable strategy in this population. This model can detect and treat asymptomatic cases of chlamydia and avoid many of the barriers associated with testing for sexually transmitted infections (STIs) in clinical settings.

Related:

• Telling partners about chlamydia – how ?
• Chlamydia Infection
• UK chlamydia cases up in under-16s
• Low uptake of Chlamydia screening in the UK

Given the critical role of interferons (IFNs) as a first line of defense against infection, it is not surprising that many viruses have evolved strategies to block an IFN response as a means to increase their replication efficiency. Virus-mediated inhibition of IFNs can be generalized into three categories, including disruption of IFN induction, disruption of IFN-inducible signaling and disruption of IFN-mediated effector functions.

The non-structural protein 1 (NS1) of influenza A viruses exerts its inhibitory effects on IFN predominately by interfering with IFN productionType I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β.

Expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. However, treatment of ex vivo human lung tissues with IFN-α results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections.

Influenza Virus Non-Structural Protein 1 (NS1) Disrupts Interferon Signaling. (2010) PLoS ONE 5(11): e13927. doi:10.1371/journal.pone.0013927

Related:

• Virus tricks to grid-lock the type I interferon system
• Interferons and Viruses
• The Interferon Antiviral Response

The types of gut bacteria that populate the guts of primates depend on the species of the host as well as where the host lives and what they eat. A new study examines the gut microbial communities in great apes, showing that a host’s species, rather than their diet, has the greatest effect on gut bacteria diversity.

Bacteria are crucial to human health. They enhance the immune system, protect against toxins, and assist in the maturation and renewal of intestinal cells. Gut microbes outnumber our own cells by 10 to 1 but little is known about how certain species come to populate our stomachs, which are sterile at birth. What causes this variation within microbial communities has been a matter of debate. Some scientists have argued that diet and habitat play the most prominent roles. The new research finds that diversity in the composition of these gut communities, not including those occasional transients and unwelcome visitors such as pathogenic bacteria, depends primarily upon the host species.

Using genetic markers, the researchers measured the diversity and abundance of various microbial species found in fecal matter of five great ape species collected in their native ranges and discovered that bacterial populations assorted to species. Moreover, the relationships of the microbial communities matched that of their host. In other words, not only is it possible to differentiate chimpanzees from humans by examining the microbial populations within their guts, but these gut microbes have been tracking the evolution of their hosts for millions of years.

Evolutionary Relationships of Wild Hominids Recapitulated by Gut Microbial Communities. (2010) PLoS Biol 8(11): e1000546. doi:10.1371/journal.pbio.1000546
Multiple factors over the lifetime of an individual, including diet, geography, and physiologic state, will influence the microbial communities within the primate gut. To determine the source of variation in the composition of the microbiota within and among species, we investigated the distal gut microbial communities harbored by great apes, as present in fecal samples recovered within their native ranges. We found that the branching order of host-species phylogenies based on the composition of these microbial communities is completely congruent with the known relationships of the hosts. Although the gut is initially and continuously seeded by bacteria that are acquired from external sources, we establish that over evolutionary timescales, the composition of the gut microbiota among great ape species is phylogenetically conserved and has diverged in a manner consistent with vertical inheritance.

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• Human gut microbiota in obesity and after gastric bypass
• Gut Feeling
• Do gut bacteria cause cancer?
• Antibiotics, your gut, and you

Microbiologists at the University of Leicester have worked on Streptococcus pneumoniae (pneumococcus) for many years. This research is now starting to pave the way for more effective vaccines against this pathogen. The pneumococcus is a pathogen of global significance, responsible for millions of deaths annually from pneumonia, meningitis and septicaemia while also causing other less serious infections, such as otitis media and sinusitis. In order to develop improved pneumococcal vaccines it is essential to understand how the bacterium interacts with the host immune system.

Pneumococci produce a range of pathogenicity factors, among which the toxin pneumolysin plays a central role and has potential as a vaccine candidate. A new paper demonstrates that pneumolysin can directly activate innate immune cells and dramatically amplify the production of pro-inflammatory cytokines. These enhancing effects of the toxin do not require Toll-like receptor (TLR)4. In particular, the toxin exerts a potent effect on interleukin (IL)-1, which is an endogenous pyrogen and powerful activator of IL-17A production. This effect results from activation of the NLRP3 inflammasome complex and NLRP3 is required for protection against the pathogen in vivo. To induce protective immunity against pneumococci, IFN-γ and IL-17A are thought to be essential. Pneumolysin plays a key role in promoting these cytokines both in vitro and in vivo during respiratory infection. The results add significantly to our understanding of the interactions between pneumococci and the immune system and support investigations into the inclusion of pneumolysin or its derivatives in novel pneumococcal vaccines.

Pneumolysin Activates the NLRP3 Inflammasome and Promotes Proinflammatory Cytokines Independently of TLR4. (2010) PLoS Pathog 6(11): e1001191. doi:10.1371/journal.ppat.1001191
Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system.

Related:

• Specific pneumococcus serotypes associated with increased mortality
• New Insights Could Lead to a Better Pneumococcal Vaccine
• Pneumococcus: the quorum-sensing killer

At least 15 million doses of anti-rabies post-exposure prophylaxis are administered annually worldwide, and an estimated 55,000 people die of rabies every year. Over 99% of these deaths occur in developing countries, predominantly in Asia and in Africa where rabies is endemic in domestic dogs. Despite the global health burden due to rabies, little is known about the patterns of the spread of dog rabies in these endemic regions. A recent paper examines the dynamics and determinants of the spatial diffusion of dog rabies viruses in North Africa based on virus genetic data. This analysis reveals a combination of restricted spread across administrative borders, the occasional long-distance movement of rabies viruses, and a strong fit between spatial spread of the virus and road distances between localities. Together, these data indicate that by transporting dogs, humans have played a key role in the dispersal of a major animal pathogen. This study provides essential new information on the transmission dynamics of rabies in Africa and will greatly assist in future intervention strategies.

Phylodynamics and Human-Mediated Dispersal of a Zoonotic Virus. (2010) PLoS Pathog 6(10): e1001166. doi:10.1371/journal.ppat.1001166
Understanding the role of humans in the dispersal of predominately animal pathogens is essential for their control. We used newly developed Bayesian phylogeographic methods to unravel the dynamics and determinants of the spread of dog rabies virus (RABV) in North Africa. Each of the countries studied exhibited largely disconnected spatial dynamics with major geo-political boundaries acting as barriers to gene flow. Road distances proved to be better predictors of the movement of dog RABV than accessibility or raw geographical distance, with occasional long distance and rapid spread within each of these countries. Using simulations that bridge phylodynamics and spatial epidemiology, we demonstrate that the contemporary viral distribution extends beyond that expected for RABV transmission in African dog populations. These results are strongly supportive of human-mediated dispersal, and demonstrate how an integrated phylogeographic approach will turn viral genetic data into a powerful asset for characterizing, predicting, and potentially controlling the spatial spread of pathogens.

Related:

• Transmission and elimination of rabies
• Pre-exposure and Post-exposure Prevention of rabies
• World Rabies Day, 28 September