MicrobiologyBytes

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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Inflammatory responses generated during virus infections are critical for antiviral immune responses. The exact virus recognition elements that activate cells to induce pro-inflammatory signals are not completely characterized. Virus recognition elements such as dsRNA and 5′-triphosphate single-stranded RNA are recognized by several cellular pathways. The intracellular or extracellular interaction of cells with virus recognition elements results in activation of innate immune responses as indicated by expression of inflammatory cytokines and chemokines. In addition to the innate immune inflammation, virus recognition elements trigger establishment of an antiviral state, under which each cell resists virus infection. The resistance to virus infection is in part through inhibition of virus replication by perturbation of RNA and protein synthesis. Determining the exact mechanisms by which immune and antivirus responses are activated is essential for understanding virus pathogenesis.

RNA with high inosine content is not commonly found in normally growing eukaryotic cells but it is present during infections with DNA and RNA viruses such as polyomavirus, Rous-associated virus, vesicular stomatitis virus, measles virus, and respiratory syncytial virus. Extracellular Ino-RNA is generated during virus infections. Cell lysis occurs frequently during virus infections, which results in the release of cell content, including intracellular generated Ino-RNA, into the extracellular space. Extracellular dsRNA has been shown to be able to stimulate antiviral responses in neighboring, uninfected cells.

Using RSV infection as a model, this paper reports that the presence of inosines in ssRNA is a potent inducer of inflammatory cytokines and the antiviral state during virus infection and suggests that Ino-RNA, of virus or cellular origin, in the surrounding tissue after release from infected cells is a signal for the presence of virus infections.

Inosine-Containing RNA Is a Novel Innate Immune Recognition Element and Reduces RSV Infection. (2011) PLoS ONE 6(10): e26463. doi:10.1371/journal.pone.0026463
During viral infections, single- and double-stranded RNA (ssRNA and dsRNA) are recognized by the host and induce innate immune responses. The cellular enzyme ADAR-1 (adenosine deaminase acting on RNA-1) activation in virally infected cells leads to presence of inosine-containing RNA (Ino-RNA). Here we report that ss-Ino-RNA is a novel viral recognition element. We synthesized unmodified ssRNA and ssRNA that had 6% to16% inosine residues. The results showed that in primary human cells, or in mice, 10% ss-Ino-RNA rapidly and potently induced a significant increase in inflammatory cytokines, such as interferon (IFN)-β (35 fold), tumor necrosis factor (TNF)-α (9.7 fold), and interleukin (IL)-6 (11.3 fold) (p

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Flagella-driven bacterial motility, referred to as swimming, has been recognized for over 20 years to affect the ability of bacteria to infect and colonize a host. The common theme is that bacteria must be motile to colonize the host but must become non-motile to chronically persist; this has been observed in many pathogenic bacteria including species of Vibrio and Pseudomonas. Therefore it makes sense that the immune system would evolve mechanisms to exploit this virulence determinant of pathogenic bacteria. This paper presents evidence that flagellar motility is recognized by innate immune cells as a phagocytic activation signal. It shows that step-wise loss of flagellar motility confers a proportional ability to evade phagocytic engulfment, independent of the flagellum itself acting as a phagocytic activator. This is not due to motility- co-regulated secretions or compensatory genetic changes by the bacteria, but instead is due to a mechano-sensory response whereby phagocytic cells respond directly to flagellar motility. This represents a novel mechanism by which the innate immune system facilitates clearance of bacterial pathogens, and provides an explanation for how selective pressure may result in bacteria with down-regulated flagellar gene expression and motility as is observed in isolates taken from chronic infections.

Step-Wise Loss of Bacterial Flagellar Torsion Confers Progressive Phagocytic Evasion. (2011) PLoS Pathog 7(9): e1002253. doi:10.1371/journal.ppat.1002253
Phagocytosis of bacteria by innate immune cells is a primary method of bacterial clearance during infection. However, the mechanisms by which the host cell recognizes bacteria and consequentially initiates phagocytosis are largely unclear. Previous studies of the bacterium Pseudomonas aeruginosa have indicated that bacterial flagella and flagellar motility play an important role in colonization of the host and, importantly, that loss of flagellar motility enables phagocytic evasion. Here we use molecular, cellular, and genetic methods to provide the first formal evidence that phagocytic cells recognize bacterial motility rather than flagella and initiate phagocytosis in response to this motility. We demonstrate that deletion of genes coding for the flagellar stator complex, which results in non-swimming bacteria that retain an initial flagellar structure, confers resistance to phagocytic binding and ingestion in several species of the gamma proteobacterial group of Gram-negative bacteria, indicative of a shared strategy for phagocytic evasion. Furthermore, we show for the first time that susceptibility to phagocytosis in swimming bacteria is proportional to mot gene function and, consequently, flagellar rotation since complementary genetically- and biochemically-modulated incremental decreases in flagellar motility result in corresponding and proportional phagocytic evasion. These findings identify that phagocytic cells respond to flagellar movement, which represents a novel mechanism for non-opsonized phagocytic recognition of pathogenic bacteria.

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A number of flaviviruses constitute a significant threat to global health. Dengue virus (DENV) infection causes around 21,000 human deaths annually, and it is estimated that at least 120 countries have endemic DENV transmission, whilst in recent years, West Nile virus (WNV) has become more prominent as a zoonotic agent, particularly in North America where the virus first emerged in 1999 and rapidly spread across the continent. WNV has now emerged in a number of European countries, particularly around the Mediterranean basin, where infections in humans, horses and birds have been reported.

Cross-reactivity of sera raised against one flavivirus recognising another flavivirus has been well documented. One consequence of flavivirus cross-reactivity is the occurrence of false-positive results, yet cross-reactivity can lead to cross-protection. Understanding and manipulating the cross-reactive properties of flaviviruses has the potential to assist the development of effective broad-spectrum human vaccines against WNV and other existing and emerging flaviviruses.

Flavivirus-induced antibody cross-reactivity. J Gen Virol. Sep 7 2011
Dengue viruses (DENV) cause countless human deaths each year, whilst West Nile virus (WNV) has re-emerged as an important human pathogen. There are currently no WNV or DENV vaccines licensed for human use, yet vaccines exist against other flaviviruses. To investigate flavivirus cross-reactivity, sera from a human cohort with a history of vaccination against tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV) and yellow fever virus (YFV) were tested for antibodies by plaque reduction neutralisation test. Neutralisation of Louping ill virus (LIV) occurred, but no significant neutralisation of Murray Valley encephalitis virus (MVEV) was observed. Sera from some individuals vaccinated against TBEV and JEV neutralised WNV, which was enhanced by YFV vaccination in some recipients. Similarly, some individuals neutralised DENV-2, but this was not significantly influenced by YFV vaccination. Antigenic cartography techniques were used to generate a geometric illustration of the neutralisation titres of selected sera against WNV, TBEV, JEV, LIV, YFV and DENV-2. This demonstrated the individual variation in antibody responses. Most sera had detectable titres against LIV and some had titres against WNV and DENV-2. Generally, LIV titres were similar to titres against TBEV, confirming the close antigenic relationship between TBEV and LIV. JEV was also antigenically closer to TBEV than WNV, using these sera. The use of sera from individuals vaccinated against multiple pathogens is unique relative to previous applications of antigenic cartography techniques. It is evident from these data that notable differences exists between amino acid sequence identity and mapped antigenic relationships within the family Flaviviridae.

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Pseudomonas aeruginosa is a common bacterium that can infect and cause disease in a wide variety of hosts, ranging from humans to plants. In healthy individuals, the innate immune system can counteract this microorganism effectively; however immunocompromised patients and cystic fibrosis patients suffer from severe infections with this bacterium. P. aeruginosa can propel itself through tissue by rotation of its long tail, called the flagellum, which is essential to establish colonization and infection of the host. The building blocks of the bacterial flagellum are over a thousand copies of the highly conserved protein flagellin. Mammals and plants have developed recognition systems to detect many different bacteria by sensing flagellin via Toll-like receptor 5 and Flagellin. Bacteria actively try to interfere with this recognition (immune evasion).

This new study describes a novel mechanism of P. aeruginosa to escape flagellin recognition. The secreted protein alkaline protease of P. aeruginosa, degrades immunity activating free flagellin. Bacterial motility is maintained, because flagellin present as building block of flagella is not degraded. In this way, the bacterium impairs recognition and hides itself from destruction by the immune system. Understanding these immune evasion strategies is of extreme importance for the development of new therapeutic approaches.

Pseudomonas Evades Immune Recognition of Flagellin in Both Mammals and Plants. 2011 PLoS Pathog 7(8): e1002206. doi:10.1371/journal.ppat.1002206
The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host innate immune systems. Recognition of flagellin monomers occurs by flagellin-specific pattern-recognition receptors, such as Toll-like receptor 5 (TLR5) in mammals and flagellin-sensitive 2 (FLS2) in plants. Activation of these immune systems via flagellin leads eventually to elimination of the bacterium from the host. In order to prevent immune activation and thus favor survival in the host, bacteria secrete many proteins that hamper such recognition. In our search for Toll like receptor (TLR) antagonists, we screened bacterial supernatants and identified alkaline protease (AprA) of Pseudomonas aeruginosa as a TLR5 signaling inhibitor as evidenced by a marked reduction in IL-8 production and NF-κB activation. AprA effectively degrades the TLR5 ligand monomeric flagellin, while polymeric flagellin (involved in bacterial motility) and TLR5 itself resist degradation. The natural occurring alkaline protease inhibitor AprI of P. aeruginosa blocked flagellin degradation by AprA. P. aeruginosa aprA mutants induced an over 100-fold enhanced activation of TLR5 signaling, because they fail to degrade excess monomeric flagellin in their environment. Interestingly, AprA also prevents flagellin-mediated immune responses (such as growth inhibition and callose deposition) in Arabidopsis thaliana plants. This was due to decreased activation of the receptor FLS2 and clearly demonstrated by delayed stomatal closure with live bacteria in plants. Thus, by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition by the innate immune systems of both mammals and plants.

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The complement system is a major component of innate immunity and consists of both soluble factors and cell surface receptors that interact to sense and respond to invading pathogens. The complement system links the innate and adaptive immune responses by a variety of mechanisms including enhancing humoral immunity, regulating antibody effector mechanisms, and modulating T cell function. In addition to these roles in normal host immune responses, the complement system has pathogenic roles in a variety of ischemic, inflammatory, and autoimmune diseases.

The complement system is a critical determinant of the outcome of infection by a variety of different viruses. Our understanding of the mechanisms by which complement protects from virus-induced disease has improved dramatically. Research in this area will not only continue to contribute to our knowledge of viral pathogenesis, but will continue to provide insight into the regulation of immune responses, and lead to improved therapeutic and vaccine approaches for both viral and non-viral pathogens. Perhaps less well understood are the mechanisms by which complement functions as a pathogenic effector in some virus-induced diseases. Further progress towards identifying the signals and pathways that lead to complement activation, which are not understood for many viruses, particularly in vivo, and a deeper understanding of the impact of complement activation on host immune responses to viral infection may shed light. Continued investigation of the role of complement in viral pathogenesis will provide important insights into virus–host interactions and strategies to prevent or treat virus-induced disease.

Complement and viral pathogenesis. Virology. 2011 411(2): 362-373
The complement system functions as an immune surveillance system that rapidly responds to infection. Activation of the complement system by specific recognition pathways triggers a protease cascade, generating cleavage products that function to eliminate pathogens, regulate inflammatory responses, and shape adaptive immune responses. However, when dysregulated, these powerful functions can become destructive and the complement system has been implicated as a pathogenic effector in numerous diseases, including infectious diseases. This review highlights recent discoveries that have identified critical roles for the complement system in the pathogenesis of viral infection.

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The main impediment to a cure for HIV is the existence of long-lasting treatment resistant virus reservoirs. This review discusses what is currently known about reservoirs, including their formation and maintenance, while focusing on latently infected CD4+ T cells. It compares several different in vivo and in vitro models of latency and comments on how each model may reflect the properties of reservoirs in vivo, especially with regard to cell phenotype, since recent studies demonstrate that multiple CD4+ T cell subsets contribute to HIV reservoirs and that with HAART and disease progression the relative contribution of different subsets may change. It also focuses on the direct infection of resting CD4+ T cells as a source of reservoir formation and as a model of latency, since recent results help explain the misconception that resting CD4+ T cells appeared to be resistant to HIV in vitro.

HIV reservoirs and latency models. Virology. 2011 411(2): 344-354

The complement system functions as an immune surveillance system that rapidly responds to infection. Activation of the complement system by specific recognition pathways triggers a protease cascade, generating cleavage products that function to eliminate pathogens, regulate inflammatory responses, and shape adaptive immune responses. However, when dysregulated, these powerful functions can become destructive and the complement system has been implicated as a pathogenic effector in numerous diseases, including infectious diseases. This review highlights recent discoveries that have identified critical roles for the complement system in the pathogenesis of viral infection.

Complement and viral pathogenesis. Virology. 2011 411(2): 362-373