MicrobiologyBytes

During the past decade, there has been a striking increase in Clostridium difficile infections worldwide predominantly due to the emergence of epidemic or hypervirulent isolates, leading to an increased research focus on this bacterium. Particular interest has surrounded the two large clostridial toxins encoded by most virulent isolates, known as toxin A and toxin B. Toxin A was thought to be the major virulence factor for many years; however, it is becoming increasingly evident that toxin B plays a much more important role than anticipated. It is clear that further experiments are required to accurately determine the relative roles of each toxin in disease, especially in more clinically relevant current epidemic isolates.

The role of toxin A and toxin B in the virulence of Clostridium difficile. Trends in Microbiology, 7 December 2011

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Listeria monocytogenes is responsible for the severe foodborne disease listeriosis. During pathogenesis, invasion of nonphagocytic cells by L. monocytogenes is crucial for crossing the host epithelial barriers and colonization of multiple organs including the liver. A new study investigated the role of the pore-forming toxin listeriolysin O (LLO) in L. monocytogenes entry into human hepatocytes.

LLO belongs to the largest family of bacterial pore-forming toxins called the cholesterol-dependent cytolysins and is a major virulence factor of L. monocytogenes. The research showed that LLO is required for efficient entry of L. monocytogenes into hepatocytes and shed light on the molecular processes involved in this activity. LLO induces tyrosine kinase(s), dynamin, and F-actin-dependent formation of an internalization vesicle. Similarly to LLO, the pore-forming toxin pneumolysin regulates bacterial entry into host cells. These findings indicate that host membrane perforation by a pore-forming toxin can be used as an invasion strategy by L. monocytogenes and raises the hypothesis that other bacteria may use a similar entry pathway.

The Pore-Forming Toxin Listeriolysin O Mediates a Novel Entry Pathway of L. monocytogenes into Human Hepatocytes. (2011)PLoS Pathog 7(11): e1002356. doi:10.1371/journal.ppat.1002356
Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.

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The native conformations of viruses and toxins are assembled to withstand harsh extracellular environments, yet they efficiently disassemble upon engaging a host cell. These reactions invariably allow the virus and toxin to gain host entry. How a stably assembled virus or toxin unravels as it encounters a host cell is remarkable. What driving force harbored in a host cell untangles the numerous covalent and noncovalent forces holding these toxic agents together? What precise function does disassembly serve?

Many viruses and toxins disassemble to enter host cells and cause disease. These conformational changes must be orchestrated temporally and spatially during entry to avoid premature disassembly leading to nonproductive pathways. Although viruses and toxins are evolutionarily distinct toxic agents, emerging findings in their respective fields have revealed that the cellular locations supporting disassembly, the host factors co-opted during disassembly, the nature of the conformational changes, and the physiological function served by disassembly are strikingly conserved. This review examines some of the shared disassembly principles observed in model viruses and toxins. Where appropriate, it underscores their differences, with the intention to draw together the fields of virus and toxin cell entry by using lessons gleaned from each field to inform and benefit one another.

How Viruses and Toxins Disassemble to Enter Host Cells. (2011) Annual Review of Microbiology 65: 287-305 doi: 10.1146/annurev-micro-090110-102855

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Researchers have discovered a toxin – SElX – released by methicillin-resistant Staphylococcus aureus (MRSA) which leads the body’s immune system to go into overdrive and damage healthy cells. SElX is made by 95 per cent of S. aureus strains, making it a potential drug target to fight this hospital superbug. SElX belongs to a family of toxins known as superantigens that can invoke an extreme immune response. When it is released it triggers an over multiplication of immune cells, which can lead to high fever, toxic shock and potentially fatal lung infections looked at a strain of MRSA known as USA300 that can cause severe infections in otherwise healthy individuals. If we can find ways to target this toxin, we may be able to stop it from triggering an over-reaction of the body’s immune system and prevent severe infections.

A Novel Core Genome-Encoded Superantigen Contributes to Lethality of Community-Associated MRSA Necrotizing Pneumonia. (2011) PLoS Pathog 7(10): e1002271. doi:10.1371/journal.ppat.1002271

Other research has linked a naturally occurring mutation in the bacterium Clostridium difficile to severe and debilitating diarrhoea in hospital patients undergoing antibiotic therapy. These antibiotics destroy the “good” bacteria in the gut, which allows this “bad” bacterium to colonise the colon, where it causes bowel infections that are difficult to treat. The mutation wipes out an inbuilt disease regulator, called anti-sigma factor TcdC, producing hypervirulent strains of C. difficile that are resistant to antibiotics and which have been found to circulate in Canada, the US, UK, Europe and Australia. The results suggest that bacterial strains carrying this mutation have the potential to produce more of the harmful toxins that cause disease in susceptible individuals – commonly patients aged 65 years or over. As we now have a better understanding of these strains, we can design new strategies to prevent, control and treat these infections.

The Anti-Sigma Factor TcdC Modulates Hypervirulence in an Epidemic BI/NAP1/027 Clinical Isolate of Clostridium difficile. (2011) PLoS Pathog 7(10): e1002317. doi:10.1371/journal.ppat.1002317

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To reach the central nervous system (CNS), pathogens have to circumvent the wall of tightly sealed endothelial cells that compose the blood–brain barrier. Neuronal projections that connect to peripheral cells and organs are the Achilles heels in CNS isolation. Some viruses and bacterial toxins interact with membrane receptors that are present at nerve terminals to enter the axoplasm. Pathogens can then be mistaken for cargo and recruit trafficking components, allowing them to undergo long-range axonal transport to neuronal cell bodies. This review highlights the strategies used by pathogens to exploit axonal transport during CNS invasion.

A hitchhiker’s guide to the nervous system: the complex journey of viruses and toxins. 2010 Nature Reviews Microbiology 8: 645-655 doi:10.1038/nrmicro2395

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• Bacterial toxins

The Shiga toxin family, a group of structurally and functionally related exotoxins, includes Shiga toxin from Shigella dysenteriae serotype 1 and the Shiga toxins that are produced by enterohaemorrhagic Escherichia coli (EHEC) strains. The existence of different, interchangeable terms to describe very similar toxins has historical reasons. The Japanese microbiologist Kiyoshi Shiga was the first to characterize the bacterial origin of dysentery caused by S. dysenteriae, in 1897. In 1977, Konowalchuk discovered a group of E. coli isolates that produced a factor that was able to kill Vero cells in culture. The factor was termed verotoxin, and the bacteria were termed verotoxin-producing E. coli (VTEC). O’Brien and colleagues recognized in the early 1980s that some E. coli isolates produced a toxin that was related to Shiga toxin and named these organisms Shiga-like toxin-producing E. coli (STEC). In 1983, it was recognized that STEC strains are associated with haemolytic uraemic syndrome (HUS). Researchers eventually realized that they were studying identical or highly related toxins.

Shiga toxin is the prototype of the Shiga toxin family and nearly identical to the E. coli-produced Shiga toxin 1 (Stx1), differing by a single amino acid. Severe disease has been epidemiologically linked to the presence of Stx2. Although Stx1 and Stx2 share a common receptor and possess the same intracellular mechanism of action, they are immunologically distinct and only 56% identical at the amino acid sequence level.

These toxins have received considerable attention not only from microbiologists but also in the field of cell biology, where it has become a powerful tool to study intracellular trafficking. In this Review, we summarize the Shiga toxin family members and their structures, receptors, trafficking pathways and cellular targets. This review discusses how Shiga toxin affects cells not only by inhibiting protein biosynthesis but also through the induction of signalling cascades that lead to apoptosis. It also discusses how Shiga toxins might be exploited in cancer therapy and immunotherapy.

Shiga toxins – from cell biology to biomedical applications. 2010 Nature Reviews Microbiology 8: 105-116. doi: 10.1038/nrmicro2279

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• Bacterial toxins
• The continuing evolution of E. coli O157:H7
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Millions of years of coevolution of plants and microbial pathogens have shaped both the abilities of microbial pathogens to overcome plant disease resistance and the abilities of plants to cope with microbial invasion. Phytopathogens from different taxonomic origins secrete structurally unrelated effectors into plants to establish infection and to suppress host defences. In addition, phytopathogenic micro-organisms produce a wide range of cytolytic toxins that function as virulence determinants.

Microbial pattern recognition is a prerequisite for the initiation of antimicrobial defenses in all multicellular organisms, including plants. The bipartite plant immune system is based upon recognition of pathogen-associated molecular patterns by pattern-recognition receptors as well as upon the activities of resistance proteins that have evolved to recognize the presence or activities of microbial effectors. In addition to the recognition of microbial patterns and effectors, plants also possess capacities to sense host-derived damage patterns that originate, for example, from the degradation of the plant cell wall by microbial hydrolytic enzymes.

Paradoxically, some phytopathogenic microbe-derived cytolytic toxins have also been reported to elicit plant defences. However, for virtually all microbial toxins with plant defence-stimulating potential, it is unknown whether activation of plant defences results from toxin-induced cellular distress or, independently of toxin action, from recognition of toxins as microbial patterns by plant pattern-recognition receptors.

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NLPs are a superfamily of proteins that are produced by various phytopathogenic micro-organisms, both prokaryotes and eukaryotes. Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) trigger leaf necrosis that is genetically distinct from immunity-associated programmed cell death and stimulate immunity associated defences in all dicotyledonous plants tested, but not in monocotyledons such as grasses. Hence, NLPs were proposed to have dual functions in plant pathogen interactions, acting both as triggers of immune responses and as toxin-like virulence factors. The broad taxonomic distribution of NLPs, in particular their occurrence in both prokaryotic and eukaryotic species, is unusual for known microbial phytotoxins, the production of which is restricted to a narrow range of microbial species.

Recent work has determined the crystal structure of an NLP from a phytopathogenic fungus (A common toxin fold mediates microbial attack and plant defense. PNAS USA June 11 2009, doi: 10.1073/pnas.0902362106). Computational modeling of the three-dimensional structure of NLPs from another fungus and from a phytopathogenic bacterium reveals a high degree of conservation. Expression of the fungus NLPs in an NLP-deficient phytopathogenic bacteria restored bacterial virulence.

Mutation analysis revealed that identical structural properties were required to cause plasma membrane permeabilization and cytolysis in plant cells, as well as to restore bacterial virulence. The conclusion is that NLPs are conserved virulence factors whose wide taxonomic distribution is exceptional for microbial phytotoxins, and that contribute to host infection by plasma membrane destruction and cytolysis. Phytotoxin-induced cellular damage-associated activation of plant defenses is reminiscent of microbial toxin-induced inflammatory activation in vertebrates and may constitute another conserved element in animal and plant innate immunity.

Related:

• Toll-Like Receptors
• Bacterial toxins

Vibrio cholerae causes cholera, a severe diarrhoea that may lead to fatal dehydration if not treated. Results of a new study suggest that parasitic infection could reduce immunity to future cholera infection and may compromise the effectiveness of cholera vaccines. V. cholerae infections cause an estimated 5 million cases of cholera annually worldwide, primarily in impoverished areas with poor sanitation. Intestinal parasites, such as the worms called helminths, are also common in developing areas when cholera is endemic, but there has been little investigation into the impact of infection with both types of pathogen.

Cholera occurs mostly in impoverished areas where there is poor sanitation and intestinal parasites are also common. However, little is known about the relationship between intestinal parasites and cholera. To learn about how parasites affect the immune response to V. cholerae, a newly published article describes 361 patients with cholera, including 53 who had intestinal parasitic infection. It was found that cholera patients with parasitic worms had decreased antibody response to cholera toxin. The decrease was greatest in IgA antibodies, which are secreted in the intestine. However, patients with worm infection did not have a difference in their immune response to lipopolysaccharide, a sugar-based molecule that is important for immunity. These different effects on the immune response to cholera toxin and lipopolysaccharide could be explained by the effect of parasitic infection on CD4+ T cells, a type of cell that influences the development of the antibody response to proteins such as cholera toxin but may not always influence the response to sugar-based molecules.

It has been a puzzle as to why cholera vaccines that initially look so promising in trials in volunteers in Europe and the United States have been much less effective in inducing a strong immune system response in countries where cholera occurs. This study supports the idea that co-infection with intestinal worms may be part of the explanation for that discrepancy. Although additional studies are needed to understand the reason for the association between helminths and decreased immune responses to cholera, this study shows that deworming programs could have an added benefit, especially in countries where cholera is present.

Immunologic Responses to Vibrio cholerae in Patients Co-Infected with Intestinal Parasites in Bangladesh. 2009 PLoS Negl Trop Dis 3(3): e403
Infection with intestinal helminths is common and may contribute to the decreased efficacy of Vibrio cholerae vaccines in endemic compared to non-endemic areas. However, the immunomodulatory effects of concomitant intestinal parasitic infection in cholera patients have not been systematically evaluated. We evaluated V. cholerae-specific immune responses in a cohort of patients with severe cholera. 361 patients completed 21 days of observation and 53 (15%) had evidence of a concomitant intestinal parasitic infection based on direct microscopy. Although there were no significant differences in the vibriocidal or lipopolysaccharide (LPS)-specific immune responses to V. cholerae, helminth-infected cholera patients had decreased fecal and serum IgA immune responses to the B subunit of cholera toxin (CTB) as well as a more modest decrease in serum IgG response to CTB. These findings remained significant for all classes of helminth infection and when controlling for potential confounding variables such as age and nutritional status. Although we hypothesized the differential effect on CTB and LPS immune responses was due to T-cell dependent immunomodulatory effects of helminth infection, we did not find additional evidence to support a classic Th1 or Th2 polarization of the immune response to V. cholerae infection related to parasite infection. The finding that helminth infection has a profound association with the mucosal humoral immune response to V. cholerae has implications for the development of protective immunity in cholera-endemic areas and provides an additional basis for deworming programs in cholera-endemic areas. Additional studies, including further characterization of the role of T cells in the immune response to human V. cholerae infection and the development of an animal model of co-infection, may provide additional insight into the mechanisms underlying these findings.

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• Cracking the cholera code
• Bacterial toxins
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