New levels of sophistication in the transcriptional landscape of bacteria

Previously unanticipated roles of noncoding and antisense RNAs give the regulation of gene expression in bacteria an extra layer of complexity

A reverse vaccinology approach identifies putative vaccination targets in the zoonotic nematode Ascaris

Ascariasis is the most prevalent helminthic disease affecting both humans and pigs and is caused by the roundworms Ascaris lumbricoides and Ascaris suum. While preventive chemotherapy continues to be the most common control method, recent reports of anthelminthic resistance highlight the need for development of a vaccine against ascariasis. The aim of this study was to use a reverse vaccinology approach to identify potential vaccine candidates for Ascaris. Three Ascaris proteomes predicted from whole-genome sequences were analyzed. Candidate proteins were identified using open-access bioinformatic tools (e.g., Vacceed, VaxiJen, Bepipred 2.0) which test for different characteristics such as sub-cellular location, T-cell and B-cell molecular binding, antigenicity, allergenicity and phylogenetic relationship with other nematode proteins. From over 100,000 protein sequences analyzed, four transmembrane proteins were predicted to be non-allergen antigens and potential vaccine candidates. The four proteins are a Piezo protein, two voltage-dependent calcium channels and a protocadherin-like protein, are all expressed in either the muscle or ovaries of both Ascaris species, and all contained high affinity epitopes for T-cells and B-cells. The use of a reverse vaccinology approach allowed the prediction of four new potential vaccination targets against ascariasis in humans and pigs. These targets can now be further tested in in vitro and in vivo assays to prove efficacy in both pigs and humans

PerR controls oxidative stress defence and aerotolerance but not motility-associated phenotypes of Campylobacter jejuni

The foodborne bacterial pathogen Campylobacter jejuni is an obligate microaerophile, which is exposed to atmospheric oxygen during transmission through the food chain. Survival under aerobic conditions requires the concerted control of oxidative stress systems, which in C. jejuni are intimately connected with iron metabolism via the PerR and Fur regulatory proteins. Here we have characterised the roles of C. jejuni PerR in oxidative stress- and motility phenotypes, and its regulon at the level of transcription, protein expression and promoter interactions. Insertional inactivation of perR in the C. jejuni reference strains NCTC 11168, 81-176 and 81116 did not result in any growth deficiencies, but strongly increased survival in atmospheric oxygen conditions, and allowed growth around filter discs infused with up to 30% H2O2 (8.8 M). Expression of catalase, alkyl hydroperoxide reductase, thioredoxin reductase and the Rrc desulforubrerythrin were increased in the perR mutant, and this was mediated at the transcriptional level as shown by electrophoretic mobility shift assays of the katA, ahpC and trxB promoters using purified PerR. Differential RNA-seq analysis of a fur perR mutant allowed the identification of eight previously unknown transcription start sites of genes controlled by either Fur and/or PerR. Finally, inactivation of perR in C. jejuni did not result in reduced motility, and did not reduce killing of Galleria melonella wax moth larvae. In conclusion, PerR plays an important role in controlling oxidative stress resistance and aerobic survival of C. jejuni, but this role does not extend into control of motility and associated phenotypes

Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

Biofilms make an important contribution to survival and transmission of bacterial pathogens in the food chain. The human pathogen Campylobacter jejuni is known to form biofilms in vitro in food chain-relevant conditions, but the exact roles and composition of the extracellular matrix are still not clear. Extracellular DNA has been found in many bacterial biofilms and can be a major component of the extracellular matrix. Here we show that extracellular DNA is also an important component of the C. jejuni biofilm when attached to stainless steel surfaces, in aerobic conditions and on conditioned surfaces. Degradation of extracellular DNA by exogenous addition of DNase I led to rapid biofilm removal, without loss of C. jejuni viability. Following treatment of a surface with DNase I, C. jejuni was unable to re-establish a biofilm population within 48 h. Similar results were obtained by digesting extracellular DNA with restriction enzymes, suggesting the need for high molecular weight DNA. Addition of C. jejuni genomic DNA containing an antibiotic resistance marker resulted in transfer of the antibiotic resistance marker to susceptible cells in the biofilm, presumably by natural transformation. Taken together, this suggest that eDNA is not only an important component of C. jejuni biofilms and subsequent food chain survival of C. jejuni, but may also contribute to the spread of antimicrobial resistance in C. jejuni. The degradation of extracellular DNA with enzymes such as DNase I is a rapid method to remove C. jejuni biofilms, and is likely to potentiate the activity of antimicrobial treatments and thus synergistically aid disinfection treatments

Pan-Genomic Analysis of Clostridium botulinum Group II (Non-Proteolytic C. botulinum) Associated with Foodborne Botulism and Isolated from the Environment

The neurotoxin formed by Clostridium botulinum Group II is a major cause of foodborne botulism, a deadly intoxication. This study aims to understand the genetic diversity and spread of C. botulinum Group II strains and their neurotoxin genes. A comparative genomic study has been conducted with 208 highly diverse C. botulinum Group II strains (180 newly sequenced strains isolated from 16 countries over 80 years, 28 sequences from Genbank). Strains possessed a single type B, E, or F neurotoxin gene or were closely related strains with no neurotoxin gene. Botulinum neurotoxin subtype variants (including novel variants) with a unique amino acid sequence were identified. Core genome single-nucleotide polymorphism (SNP) analysis identified two major lineages—one with type E strains, and the second dominated by subtype B4 strains with subtype F6 strains. This study revealed novel details of population structure/diversity and established relationships between whole-genome lineage, botulinum neurotoxin subtype variant, association with foodborne botulism, epidemiology, and geographical source. Additionally, the genome sequences represent a valuable resource for the research community (e.g., understanding evolution of C. botulinum and its neurotoxin genes, dissecting key aspects of C. botulinum Group II biology). This may contribute to improved risk assessments and the prevention of foodborne botulism

Role of the Helicobacter pylori outer-membrane proteins AlpA and AlpB in colonization of the guinea pig stomach

The human gastric pathogen Helicobacter pylori expresses several putative outer-membrane proteins (OMPs), but the role of individual OMPs in colonization of the stomach by H. pylori is still poorly understood. The role of four such OMPs (AlpA, AlpB, OipA and HopZ) in a guinea pig model of H. pylori infection has been investigated. Single alpA, alpB, hopZ and oipA isogenic mutants were constructed in the guinea pig-adapted, wild-type H. pylori strain GP15. Guinea pigs were inoculated intragastrically with the wild-type strain, single mutants or a mixture of the wild-type and a single mutant in a 1: 1 ratio. Three weeks after infection, H. pylori could be isolated from stomach sections of all animals that were infected with the wild-type, the hopZ mutant or the oipA mutant, but from only five of nine (P = 0.18) and one of seven (P = 0.02) animals that were infected with the alpA or alpB mutants, respectively. The hopZ and oipA mutants colonized the majority of animals that were inoculated with the strain mixture, whereas alpA and alpB mutants could not be isolated from anim

In Silico Docking of Nematode β-Tubulins With Benzimidazoles Points to Gene Expression and Orthologue Variation as Factors in Anthelmintic Resistance

The efficacy of benzimidazole anthelmintics can vary depending on the target parasite, with Ascaris nematodes being highly responsive, and whipworms being less responsive. Anthelmintic resistance has become widespread, particularly in strongyle nematodes such as Haemonchus contortus in ruminants, and resistance has recently been detected in hookworms of humans and dogs. Past work has shown that there are multiple β-tubulin isotypes in helminths, yet only a few of these contribute to benzimidazole interactions and resistance. The β-tubulin isotypes of ascarids and soil-transmitted helminths were identified by mining available genome data, and phylogenetic analysis showed that the ascarids share a similar repertoire of seven β-tubulin isotypes. Strongyles also have a consistent pattern of four β-tubulin isotypes. In contrast, the whipworms only have two isotypes, with one of these clustering more basally and distinct from any other group. Key β-tubulin isotypes selected based on previous studies were the focus of in silico molecular docking simulations to look at the interactions with benzimidazoles. These showed that all β-tubulins had similar interactions with benzimidazoles and maintained the key bond with residue E198 in all species, indicating similar mechanisms of action. However, the interaction was stronger and more consistent in the strongyles and whipworms than it was in the ascarids. Alteration of β-tubulin isotypes with the common resistance-associated mutations originally identified in H. contortus resulted in similar interaction modeling for all species. In conclusion, ascarids, strongyles, and whipworms all have their own unique repertoire of β-tubulins, which could explain why benzimidazole resistance and susceptibility varies between these groups of parasites. These data complement recent work that has highlighted the roles of essential residues in benzimidazole drug binding and shows that there is a separation between strongyle parasites that frequently develop resistance and ascarid parasites, which have been much less prone to developing resistance

Identification of key interactions of benzimidazole resistance-associated amino acid mutations in Ascaris β-tubulins by molecular docking simulations

Ascaris species are soil-transmitted helminths that infect humans and livestock mainly in low and middle-income countries. Benzimidazole (BZ) class drugs have predominated for many years in the treatment of Ascaris infections, but persistent use of BZs has already led to widespread resistance in other nematodes, and treatment failure is emerging for Ascaris. Benzimidazoles act by binding to β-tubulin proteins and destabilising microtubules. Three mutations in the β-tubulin protein family are associated with BZ resistance. Seven shared β-tubulin isotypes were identified in Ascaris lumbricoides and A. suum genomes. Benzimidazoles were predicted to bind to all β-tubulin isotypes using in silico docking, demonstrating that the selectivity of BZs to interact with one or two β-tubulin isotypes is likely the result of isotype expression levels affecting the frequency of interaction. Ascaris β-tubulin isotype A clusters with helminth β-tubulins previously shown to interact with BZ. Molecular dynamics simulations using β-tubulin isotype A highlighted the key role of amino acid E198 in BZ-β-tubulin interactions. Simulations indicated that mutations at amino acids E198A and F200Y alter binding of BZ, whereas there was no obvious effect of the F167Y mutation. In conclusion, the key interactions vital for BZ binding with β-tubulins have been identified and show how mutations can lead to resistance in nematodes

A PAS domain-containing regulator controls flagella-flagella interactions in Campylobacter jejuni

The bipolar flagella of the foodborne bacterial pathogen Campylobacter jejuni confer motility, which is essential for virulence. The flagella of C. jejuni are post-translationally modified, but how this process is controlled is not well understood. In this work, we have identified a novel PAS-domain containing regulatory system, which modulates flagella-flagella interactions in C. jejuni. Inactivation of the cj1387c gene, encoding a YheO-like PAS6 domain linked to a helix-turn-helix domain, resulted in the generation of a tightly associated “cell-train” morphotype, where up to four cells were connected by their flagella. The morphotype was fully motile, resistant to vortexing, accompanied by increased autoagglutination, and was not observed in aflagellated cells. The Δcj1387c mutant displayed increased expression of the adjacent Cj1388 protein, which comprises of a single endoribonuclease L-PSP domain. Comparative genomics showed that cj1387c (yheO) orthologs in bacterial genomes are commonly linked to an adjacent cj1388 ortholog, with some bacteria, including C. jejuni, containing another cj1388-like gene (cj0327). Inactivation of the cj1388 and cj0327 genes resulted in decreased autoagglutination in Tween-20-supplemented media. The Δcj1388 and Δcj0327 mutants were also attenuated in a Galleria larvae-based infection model. Finally, substituting the sole cysteine in Cj1388 for serine prevented Cj1388 dimerization in non-reducing conditions, and resulted in decreased autoagglutination in the presence of Tween-20. We hypothesize that Cj1388 and Cj0327 modulate post-translational modification of the flagella through yet unidentified mechanisms, and propose naming Cj1387 the Campylobacter Flagella Interaction Regulator CfiR, and the Cj1388 and Cj0327 protein as CfiP and CfiQ, respectively

Diversity of the Genomes and Neurotoxins of Strains of Clostridium botulinum Group I and Clostridium sporogenes Associated with Foodborne, Infant and Wound Botulism.

Clostridium botulinum Group I and Clostridium sporogenes are closely related bacteria responsible for foodborne, infant and wound botulism. A comparative genomic study with 556 highly diverse strains of C. botulinum Group I and C. sporogenes (including 417 newly sequenced strains) has been carried out to characterise the genetic diversity and spread of these bacteria and their neurotoxin genes. Core genome single-nucleotide polymorphism (SNP) analysis revealed two major lineages; C. botulinum Group I (most strains possessed botulinum neurotoxin gene(s) of types A, B and/or F) and C. sporogenes (some strains possessed a type B botulinum neurotoxin gene). Both lineages contained strains responsible for foodborne, infant and wound botulism. A new C. sporogenes cluster was identified that included five strains with a gene encoding botulinum neurotoxin sub-type B1. There was significant evidence of horizontal transfer of botulinum neurotoxin genes between distantly related bacteria. Population structure/diversity have been characterised, and novel associations discovered between whole genome lineage, botulinum neurotoxin sub-type variant, epidemiological links to foodborne, infant and wound botulism, and geographic origin. The impact of genomic and physiological variability on the botulism risk has been assessed. The genome sequences are a valuable resource for future research (e.g., pathogen biology, evolution of C. botulinum and its neurotoxin genes, improved pathogen detection and discrimination), and support enhanced risk assessments and the prevention of botulism