The Molecular Architecture and Mode of Action of Clostridium perfringens ε-Toxin.

Clostridium perfringens ε-toxin has long been associated with a severe enterotoxaemia of livestock animals, and more recently, was proposed to play a role in the etiology of multiple sclerosis in humans. The remarkable potency of the toxin has intrigued researchers for many decades, who suggested that this indicated an enzymatic mode of action. Recently, there have been major breakthroughs by finding that it is a pore-forming toxin which shows exquisite specificity for cells bearing the myelin and lymphocyte protein (MAL) receptor. This review details the molecular structures of the toxin, the evidence which identifies MAL as the receptor and the possible roles of other cell membrane components in toxin binding. The information on structure and mode of action has allowed the functions of individual amino acids to be investigated and has led to the creation of mutants with reduced toxicity that could serve as vaccines. In spite of this progress, there are still a number of key questions around the mode of action of the toxin which need to be further investigated.

Is Clostridium perfringens epsilon toxin associated with multiple sclerosis?

Clostridium perfringens epsilon toxin is associated with enterotoxaemia in livestock. More recently, it is proposed to play a role in multiple sclerosis (MS) in humans. Compared to matched controls, strains of C. perfringens which produce epsilon toxin are significantly more likely to be isolated from the gut of MS patients and at significantly higher levels; similarly, sera from MS patients are significantly more likely to contain antibodies to epsilon toxin. Epsilon toxin recognises the myelin and lymphocyte (MAL) protein receptor, damaging the blood-brain barrier and brain cells expressing MAL. In the experimental autoimmune encephalomyelitis model of MS, the toxin enables infiltration of immune cells into the central nervous system, inducing an MS-like disease. These studies provide evidence that epsilon toxin plays a role in MS, but do not yet fulfil Koch's postulates in proving a causal role.

Molecular detection of blood-borne agents in vampire bats from Brazil, with the first molecular evidence of Neorickettsia sp. in Desmodus rotundus and Diphylla ecaudata.

Bats (Mammalia, Chiroptera) represent the second largest group of mammals. Due to their ability to fly and adapt and colonize different niches, bats act as reservoirs of several potentially zoonotic pathogens. In this context, the present work aimed to investigate, using molecular techniques, the occurrence of blood-borne agents (Anaplasmataceae, Coxiella burnetii, hemoplasmas, hemosporidians and piroplasmids) in 198 vampire bats sampled in different regions of Brazil and belonging to the species Desmodus rotundus (n = 159), Diphylla ecaudata (n = 31) and Diaemus youngii (n = 8). All vampire bats liver samples were negative in PCR assays for Ehrlichia spp., Anaplasma spp., piroplasmids, hemosporidians and Coxiella burnetii. However, Neorickettsia sp. was detected in liver samples of 1.51% (3/198) through nested PCR based on the 16S rRNA gene in D. rotundus and D. ecaudata. This is the first study to report Neorickettsia sp. in vampire bats. Hemoplasmas were detected in 6.06% (12/198) of the liver samples using a PCR based on the 16S rRNA gene. The two 16S rRNA sequences obtained from hemoplasmas were closely related to sequences previously identified in vampire and non-hematophagous bats from Belize, Peru and Brazil. The genotypic analysis identified a high diversity of bat-associated hemoplasma genotypes from different regions of the world, emphasizing the need for studies on this subject, in order to better understand the mechanisms of co-evolution between this group of bacteria and their vertebrate hosts. The role of neotropical bat-associated Neorickettsia sp. and bats from Brazil in the biological cycle of such agent warrant further investigation.

MLVA and com1 genotyping of Coxiella burnetii in farmed ruminants in Great Britain.

Coxiella burnetii, the causative agent of the zoonotic disease Q fever, has been shown to be endemic in Great Britain, but information on the prevailing genomic lineages or Genomic Groups (GGs) of Coxiella burnetii is limited. The aim of this study was to genotype C. burnetii isolates from infected farmed ruminants by Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) and identify their associated Genomic Group. A total of 51 Coxiella-containing abortion samples from farmed ruminants (sheep, goats, and cattle), which were collected in Great Britain during 2013-2018, were included in the study, 34 of which returned a C. burnetii MLVA genotype. All bovine samples (n = 18), 5/7 of the ovine samples, and 3/9 of the caprine samples belonged to an MLVA cluster which we could link to the MST20 genotype of GG III, whereas 6/9 of the caprine samples and 2/7 of the ovine samples belonged to MLVA clusters which we could link to the MST33 or MST32 genotypes of GG II (7 vs 1 sample(s), respectively). We also noted that the Coxiella-specific com1 gene contained unique mutations that could genomotype isolates, i.e. assign them to a Genomic Group. In conclusion, both goats and sheep in Great Britain (from 2014 onward) were found to carry the same MLVA genotypes (MST33-like; GG II) that were linked to a human Q fever outbreak in the Netherlands. This knowledge in combination with the usage of genotyping/genomotyping methods should prove useful in future surveillance programs and in the management of outbreaks.

The increased prevalence of Vibrio species and the first reporting of Vibrio jasicida and Vibrio rotiferianus at UK shellfish sites.

Warming sea-surface temperature has led to an increase in the prevalence of Vibrio species in marine environments. This can be observed particularly in temperate regions where conditions for their growth has become more favourable. The increased prevalence of pathogenic Vibrio species has resulted in a worldwide surge of Vibriosis infections in human and aquatic animals. This study uses sea-surface temperature data around the English and Welsh coastlines to identify locations where conditions for the presence and growth of Vibrio species is favourable. Shellfish samples collected from three locations that were experiencing an increase in sea-surface temperature were found to be positive for the presence of Vibrio species. We identified important aquaculture pathogens Vibrio rotiferianus and Vibrio jasicida from these sites that have not been reported in UK waters. We also isolated human pathogenic Vibrio species including V. parahaemolyticus from these sites. This paper reports the first isolation of V. rotiferianus and V. jasicida from UK shellfish and highlights a growing diversity of Vibrio species inhabiting British waters.

Correlating Genotyping Data of Coxiella burnetii with Genomic Groups.

Coxiella burnetii is a zoonotic pathogen that resides in wild and domesticated animals across the globe and causes a febrile illness, Q fever, in humans. Several distinct genetic lineages or genomic groups have been shown to exist, with evidence for different virulence potential of these lineages. Multispacer Sequence Typing (MST) and Multiple-Locus Variable number tandem repeat Analysis (MLVA) are being used to genotype strains. However, it is unclear how these typing schemes correlate with each other or with the classification into different genomic groups. Here, we created extensive databases for published MLVA and MST genotypes of C. burnetii and analysed the associated metadata, revealing associations between animal host and human disease type. We established a new classification scheme that assigns both MST and MLVA genotypes to a genomic group and which revealed additional sub-lineages in two genomic groups. Finally, we report a novel, rapid genomotyping method for assigning an isolate into a genomic group based on the Cox51 spacer sequence. We conclude that by pooling and streamlining existing datasets, associations between genotype and clinical outcome or host source were identified, which in combination with our novel genomotyping method, should enable an estimation of the disease potential of new C. burnetii isolates.

Bacterial dormancy: A subpopulation of viable but non-culturable cells demonstrates better fitness for revival.

The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.

Campylobacter jejuni 11168H Exposed to Penicillin Forms Persister Cells and Cells With Altered Redox Protein Activity.

The formation of persister cells is one mechanism by which bacteria can survive exposure to environmental stresses. We show that Campylobacter jejuni 11168H forms persister cells at a frequency of 10-3 after exposure to 100 × MIC of penicillin G for 24 h. Staining the cell population with a redox sensitive fluorescent dye revealed that penicillin G treatment resulted in the appearance of a population of cells with increased fluorescence. We present evidence, to show this could be a consequence of increased redox protein activity in, or associated with, the electron transport chain. These data suggest that a population of penicillin G treated C. jejuni cells could undergo a remodeling of the electron transport chain in order to moderate membrane hyperpolarization and intracellular alkalization; thus reducing the antibiotic efficacy and potentially assisting in persister cell formation.

Functional redundancy of Burkholderia pseudomallei phospholipase C enzymes and their role in virulence.

Phospholipase C (PLC) enzymes are key virulence factors in several pathogenic bacteria. Burkholderia pseudomallei, the causative agent of melioidosis, possesses at least three plc genes (plc1, plc2 and plc3). We found that in culture medium plc1 gene expression increased with increasing pH, whilst expression of the plc3 gene was pH (4.5 to 9.0) independent. Expression of the plc2 gene was not detected in culture medium. All three plc genes were expressed during macrophage infection by B. pseudomallei K96243. Comparing B. pseudomallei wild-type with plc mutants revealed that plc2, plc12 or plc123 mutants showed reduced intracellular survival in macrophages and reduced plaque formation in HeLa cells. However, plc1 or plc3 mutants showed no significant differences in plaque formation compared to wild-type bacteria. These findings suggest that Plc2, but not Plc1 or Plc3 are required for infection of host cells. In Galleria mellonella, plc1, plc2 or plc3 mutants were not attenuated compared to the wild-type strain, but multiple plc mutants showed reduced virulence. These findings indicate functional redundancy of the B. pseudomallei phospholipases in virulence.

Coxiella burnetii replicates in Galleria mellonella hemocytes and transcriptome mapping reveals in vivo regulated genes.

Larvae of the greater wax moth (Galleria mellonella) are susceptible to infection with C. burnetii, an obligate intracellular bacterial pathogen. We show that bacteria are found in hemocytes after infection, and occupy vacuoles which are morphologically similar to Coxiella-containing vacuoles seen in infected mammalian phagocytes. We characterized the infection by transcriptome profiling of bacteria isolated from the hemocytes of infected larvae and identified 46 highly upregulated genes. The encoded proteins are predicted to be involved in translation, LPS biosynthesis, biotin synthesis, scavenging of reactive oxygen species, and included a T4SS effector and 30 hypothetical proteins. Some of these genes had previously been shown to be upregulated in buffalo green monkey (BGM) cells or in mice, whilst others appear to be regulated in a host-specific manner. Altogether, our results demonstrate the value of the G. mellonella model to study intracellular growth and identify potential virulence factors of C. burnetii.

Trehalose and bacterial virulence.

Trehalose is a disaccharide of two D-glucose molecules linked by a glycosidic linkage, which plays both structural and functional roles in bacteria. Trehalose can be synthesized and degraded by several pathways, and induction of trehalose biosynthesis is typically associated with exposure to abiotic stress. The ability of trehalose to protect against abiotic stress has been exploited to stabilize a range of bacterial vaccines. More recently, there has been interest in the role of this molecule in microbial virulence. There is now evidence that trehalose or trehalose derivatives play important roles in virulence of a diverse range of Gram-positive and Gram-negative pathogens of animals or plants. Trehalose and/or trehalose derivatives can play important roles in host colonization and growth in the host, and can modulate the interactions with host defense mechanisms. However, the roles are typically pathogen-specific. These findings suggest that trehalose metabolism may be a target for novel pathogen-specific rather than broad spectrum interventions.

Identification of Burkholderia pseudomallei Genes Induced During Infection of Macrophages by Differential Fluorescence Induction.

Burkholderia pseudomallei, the causative agent of melioidosis, can survive and replicate in macrophages. Little is known about B. pseudomallei genes that are induced during macrophage infection. We constructed a B. pseudomallei K96243 promoter trap library with genomic DNA fragments fused to the 5' end of a plasmid-borne gene encoding enhanced green fluorescent protein (eGFP). Microarray analysis showed that the library spanned 88% of the B. pseudomallei genome. The recombinant plasmids were introduced into Burkholderia thailandensis E264, and promoter fusions active during in vitro culture were removed. J774A.1 murine macrophages were infected with the promoter trap library, and J774A.1 cells containing fluorescent bacteria carrying plasmids with active promoters were isolated using flow cytometric-based cell sorting. Candidate macrophage-induced B. pseudomallei genes were identified from the location of the insertions containing an active promoter activity. A proportion of the 138 genes identified in this way have been previously reported to be involved in metabolism and transport, virulence, or adaptation. Novel macrophage-induced B. pseudomallei genes were also identified. Quantitative reverse-transcription PCR analysis of 13 selected genes confirmed gene induction during macrophage infection. Deletion mutants of two macrophage-induced genes from this study were attenuated in Galleria mellonella larvae, suggesting roles in virulence. B. pseudomallei genes activated during macrophage infection may contribute to intracellular life and pathogenesis and merit further investigation toward control strategies for melioidosis.

Isolation and primary culture of Galleria mellonella hemocytes for infection studies.

Galleria mellonella larvae are increasingly used to study the mechanisms of virulence of microbial pathogens and to assess the efficacy of antimicrobials.  The G. mellonella model can faithfully reproduce many aspects of microbial disease which are seen in mammals, and therefore allows a reduction in the use of mammals. The model is now being widely used by researchers in universities, research institutes and industry. An attraction of the model is the interaction between pathogen and host. Hemocytes are specialised phagocytic cells which resemble neutrophils in mammals and play a major role in the response of the larvae to infection. However, the detailed interactions of hemocytes with pathogens is poorly understood, and is complicated by the presence of different sub-populations of cells. We report here a method for the isolation of hemocytes from Galleria mellonella.  A needle-stick injury of larvae, before harvesting, markedly increased the recovery of hemocytes in the hemolymph. The majority of the hemocytes recovered were granulocyte-like cells. The hemocytes survived for at least 7 days in culture at either 28°C or 37°C. Pre-treatment of larvae with antibiotics did not enhance the survival of the cultured hemocytes. Our studies highlight the importance of including sham injected, rather than un-injected, controls when the G. mellonella model is used to test antimicrobial compounds. Our method will now allow investigations of the interactions of microbial pathogens with insect hemocytes enhancing the value of G. mellonella as an alternative model to replace the use of mammals, and for studies on hemocyte biology.

From cell culture to cynomolgus macaque: infection models show lineage-specific virulence potential of Coxiella burnetii.

Coxiella burnetii is an obligate intracellular pathogen that causes the zoonotic disease Q fever in humans, which can occur in either an acute or a chronic form with serious complications. The bacterium has a wide host range, including unicellular organisms, invertebrates, birds and mammals, with livestock representing the most significant reservoir for human infections. Cell culture models have been used to decipher the intracellular lifestyle of C. burnetii, and several infection models, including invertebrates, rodents and non-human primates, are being used to investigate host-pathogen interactions and to identify bacterial virulence factors and vaccine candidates. However, none of the models replicate all aspects of human disease. Furthermore, it is becoming evident that C. burnetii isolates belonging to different lineages exhibit differences in their virulence in these models. Here, we compare the advantages and disadvantages of commonly used infection models and summarize currently available data for lineage-specific virulence.

Clostridium perfringens epsilon toxin vaccine candidate lacking toxicity to cells expressing myelin and lymphocyte protein.

A variant form of Clostridium perfringens epsilon toxin (Y30A-Y196A) with mutations, which shows reduced binding to Madin-Darby canine kidney (MDCK) cells and reduced toxicity in mice, has been proposed as the next-generation enterotoxaemia vaccine. Here we show that, unexpectedly, the Y30A-Y196A variant does not show a reduction in toxicity towards Chinese hamster ovary (CHO) cells engineered to express the putative receptor for the toxin (myelin and lymphocyte protein; MAL). The further addition of mutations to residues in a second putative receptor binding site of the Y30A-Y196A variant further reduces toxicity, and we selected Y30A-Y196A-A168F for further study. Compared to Y30A-Y196A, Y30A-Y196A-A168F showed more than a 3-fold reduction in toxicity towards MDCK cells, more than a 4-fold reduction in toxicity towards mice and at least 200-fold reduction in toxicity towards CHO cells expressing sheep MAL. The immunisation of rabbits or sheep with Y30A-Y196A-A168F induced high levels of neutralising antibodies against epsilon toxin, which persisted for at least 1 year. Y30A-Y196A-A168F is a candidate for development as a next-generation enterotoxaemia vaccine.