Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.

Characterization of the Phase-Variable Autotransporter Lav Reveals a Role in Host Cell Adherence and Biofilm Formation in Nontypeable Haemophilus influenzae.

Lav is an autotransporter protein found in pathogenic Haemophilus and Neisseria species. Lav in nontypeable Haemophilus influenzae (NTHi) is phase-variable: the gene reversibly switches ON-OFF via changes in length of a locus-located GCAA(n) simple DNA sequence repeat tract. The expression status of lav was examined in carriage and invasive collections of NTHi, where it was predominantly not expressed (OFF). Phenotypic study showed lav expression (ON) results in increased adherence to human lung cells and denser biofilm formation. A survey of Haemophilus species genome sequences showed lav is present in ∼60% of NTHi strains, but lav is not present in most typeable H. influenzae strains. Sequence analysis revealed a total of five distinct variants of the Lav passenger domain present in Haemophilus spp., with these five variants showing a distinct lineage distribution. Determining the role of Lav in NTHi will help understand the role of this protein during distinct pathologies.

Phase-variable bacterial loci: how bacteria gamble to maximise fitness in changing environments.

Phase-variation of genes is defined as the rapid and reversible switching of expression - either ON-OFF switching or the expression of multiple allelic variants. Switching of expression can be achieved by a number of different mechanisms. Phase-variable genes typically encode bacterial surface structures, such as adhesins, pili, and lipooligosaccharide, and provide an extra contingency strategy in small-genome pathogens that may lack the plethora of 'sense-and-respond' gene regulation systems found in other organisms. Many bacterial pathogens also encode phase-variable DNA methyltransferases that control the expression of multiple genes in systems called phasevarions (phase-variable regulons). The presence of phase-variable genes allows a population of bacteria to generate a number of phenotypic variants, some of which may be better suited to either colonising certain host niches, surviving a particular environmental condition and/or evading an immune response. The presence of phase-variable genes complicates the determination of an organism's stably expressed antigenic repertoire; many phase-variable genes are highly immunogenic, and so would be ideal vaccine candidates, but unstable expression due to phase-variation may allow vaccine escape. This review will summarise our current understanding of phase-variable genes that switch expression by a variety of mechanisms, and describe their role in disease and pathobiology.

Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells.

Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-to-cell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede high-affinity protein-glycan or protein-protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the saccharide component of LOS/LPS have direct, high-affinity interactions with host glycans. Glycan microarrays reveal that LOS/LPS of four distinct bacterial pathogens bind to numerous host glycan structures. Surface plasmon resonance was used to determine the affinity of these interactions and revealed 66 high-affinity host-glycan:bacterial-glycan pairs with equilibrium dissociation constants (K(D)) ranging between 100 nM and 50 µM. These glycan:glycan affinity values are similar to those reported for lectins or antibodies with glycans. Cell assays demonstrated that glycan:glycan interaction-mediated bacterial adherence could be competitively inhibited by either host cell or bacterial glycans. This is the first report to our knowledge of high affinity glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions between a diverse range of structures suggests that these interactions may be important in all biological systems.

Phasevarions in Haemophilus influenzae biogroup aegyptius control expression of multiple proteins.

IMPORTANCE: Haemophilus influenzae biogroup aegyptius is a human-adapted pathogen and the causative agent of Brazilian purpuric fever (BPF), an invasive disease with high mortality, that sporadically manifests in children previously suffering conjunctivitis. Phase variation is a rapid and reversible switching of gene expression found in many bacterial species, and typically associated with outer-membrane proteins. Phase variation of cytoplasmic DNA methyltransferases has been shown to play important roles in bacterial gene regulation and can act as epigenetic switches, regulating the expression of multiple genes as part of systems called phasevarions (phase-variable regulons). This study characterized two alleles of the ModA phasevarion present in H. influenzae biogroup aegyptius, ModA13, found in non-BPF causing strains and ModA16, unique to BPF causing isolates. Phase variation of ModA13 and ModA16 led to genome-wide changes to DNA methylation resulting in altered protein expression. These changes did not affect serum resistance in H. influenzae biogroup aegyptius strains.

Assessment of glycan interactions of clinical and avian isolates of Campylobacter jejuni.

BACKGROUND: Campylobacter jejuni strain 11168 was demonstrated to have a broad specificity for eukaryotic surface glycosylation using glycan array analysis. The initial screen indicated that sialic acid and mannose are important binding partners after environmental stress, while galactose and fucose structures are likely to be involved in persistent infection. RESULTS: In this broader study, five additional human/clinical isolates and six chicken isolates were fully assessed to determine their glycan binding capacity using an extended glycan array. C. jejuni 11168 was rescreened here due to the presence of glycoaminoglycan (GAG) and other structures that were not available on our previous glycan array. The current array analysis of additional C. jejuni strains confirmed the growth condition dependent differences in glycan binding that was previously observed for C. jejuni 11168. We noted strain to strain variations, particularly for the human isolates C. jejuni 520 and 81116 and the chicken isolate C. jejuni 331, with the majority of differences observed in galactose, mannose and GAG binding. Chicken isolates were found to bind to a broader range of glycans compared to the human isolates, recognising branched mannose and carageenan (red seaweed) glycans. Glycan array data was confirmed using cell-based lectin inhibition assays with the fucose (UEA-I) and mannose (ConA) binding lectins. CONCLUSIONS: This study confirms that all C. jejuni strains tested bind to a broad range of glycans, with the majority of strains (all except 81116) altering recognition of sialic acid and mannose after environmental stress. Galactose and fucose structures were bound best by all strains when C. jejuni was grown under host like conditions confirming the likelihood of these structures being involved in persistent infection.

Variation of chemosensory receptor content of Campylobacter jejuni strains and modulation of receptor gene expression under different in vivo and in vitro growth conditions.

BACKGROUND: Chemotaxis is crucial for the colonisation/infection of hosts with Campylobacter jejuni. Central to chemotaxis are the group A chemotaxis genes that are responsible for sensing the external environment. The distribution of group A chemoreceptor genes, as found in the C. jejuni sequenced strains, tlp1-4, 7, 10 and 11 were determined in 33 clinical human and avian isolates. RESULTS: Group A tlp gene content varied among the strains with genes encoding tlp1 (aspartate receptor, ccaA) and tlp7 present in all strains tested, where as tlp11 was present in only one of our international collection clinical isolates, C. jejuni 520, but was more prevalent (9/13) in the freshly isolated clinical stains from patients who required hospitalisation due to C. jejuni infection (GCH1-17). Relative expression levels of the group A tlp genes were also determined in C. jejuni reference strains NCTC 11168-GS, 11168-O and 81116 using cells grown in vitro at 37°C, 42°C and maintained at room temperature and with cells isolated directly from murine and avian hosts by immune magnetic separation without subsequent culture. Gene expression of tlp genes was varied based on strain, growth conditions and in vivo isolation source. Tlp1, although the most conserved, showed the lowest and most varied mRNA expression and protein production under laboratory conditions. Tlp7 was highly expressed at most conditions tested, and gene expression was not influenced by the tlp7 gene encoding a full length protein or one expressed as separate periplasmic and cytoplasmic domains. CONCLUSION: We have shown that chemosensory receptor set variation exists among C. jejuni strains, but is not dependent on the isolation source.

Streptococcus suis pathogenesis-A diverse array of virulence factors for a zoonotic lifestyle.

Streptococcus suis is a major cause of respiratory tract and invasive infections in pigs and is responsible for a substantial disease burden in the pig industry. S. suis is also a significant cause of bacterial meningitis in humans, particularly in South East Asia. S. suis expresses a wide array of virulence factors, and although many are described as being required for disease, no single factor has been demonstrated to be absolutely required. The lack of uniform distribution of known virulence factors among individual strains and lack of evidence that any particular virulence factor is essential for disease makes the development of vaccines and treatments challenging. Here we review the current understanding of S. suis virulence factors and their role in the pathogenesis of this important zoonotic pathogen.

Actinobacillus pleuropneumoniae: The molecular determinants of virulence and pathogenesis.

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is responsible for high economic losses in swine herds across the globe. Pleuropneumonia is characterized by severe respiratory distress and high mortality. The knowledge about the interaction between bacterium and host within the porcine respiratory tract has improved significantly in recent years. A. pleuropneumoniae expresses multiple virulence factors, which are required for colonization, immune clearance, and tissue damage. Although vaccines are used to protect swine herds against A. pleuropneumoniae infection, they do not offer complete coverage, and often only protect against the serovar, or serovars, used to prepare the vaccine. This review will summarize the role of individual A. pleuropneumoniae virulence factors that are required during key stages of pathogenesis and disease progression, and highlight progress made toward developing effective and broadly protective vaccines against an organism of great importance to global agriculture and food production.

The Acinetobacter baumannii Autotransporter Adhesin Ata Recognizes Host Glycans as High-Affinity Receptors.

Acinetobacter baumannii is a significant opportunistic pathogen responsible for infections of the lung, blood, skin, urinary tract, and soft tissues, with some strains exhibiting almost complete resistance to commonly used antibiotics. This multidrug resistance, together with a dearth of new antibiotic development, mean novel methods of treatment and prevention are urgently needed. Although many A. baumannii factors required to colonize the host have been identified, little is known about the specific host molecules recognized by these factors. A. baumannii produces a trimeric autotransporter adhesin known as Ata that has been previously demonstrated to bind components of the host cell's extracellular matrix, which are often heavily glycosylated. We hypothesized that Ata would exhibit lectin activity which would play a role in adherence to the host cell surface. Our biophysical analysis using glycan arrays and surface plasmon resonance demonstrated that Ata binds galactose, N-acetylglucosamine, and galactose (ß1-3/4) N-acetylglucosamine with high-affinity. These structures are present on many of the proteins which were previously reported to be bound by Ata. We also demonstrated that the recognition of human plasma fibronectin by Ata requires this ability to bind glycans, as the interaction between Ata and fibronectin does not occur when fibronectin is deglycosylated. This strongly suggests a key role for Ata lectin activity during host adherence. This information will assist in directing the development of new and effective treatments to block host interactions using glycans and/or novel compounds in multidrug resistant A. baumannii infections.

Streptococcus suis Encodes Multiple Allelic Variants of a Phase-Variable Type III DNA Methyltransferase, ModS, That Control Distinct Phasevarions.

Streptococcus suis is a significant cause of bacterial meningitis in humans, particularly in Southeast Asia, and is a leading cause of respiratory and invasive disease in pigs. Phase-variable DNA methyltransferases, associated with restriction-modification (R-M) systems, are a source of epigenetic gene regulation, controlling the expression of multiple genes. These systems are known as phasevarions (phase-variable regulons) and have been characterized in many host-adapted bacterial pathogens. We recently described the presence of a Type III DNA methyltransferase in S. suis, ModS, which contains a simple sequence repeat (SSR) tract within the open reading frame of the modS gene and which differed in length between individual strains. We also observed that multiple allelic variants of the modS gene were present in a population of S. suis isolates. Here, we demonstrate that a biphasic ON-OFF switching of expression occurs in the two most common ModS alleles, ModS1 and ModS2, and that switching is dependent on SSR tract length. Furthermore, we show using single-molecule real-time (SMRT) sequencing that ModS1 and ModS2 are active methyltransferases in S. suis ON-OFF switching of each ModS allele results in the regulation of distinct phasevarions, with the ModS2 phasevarion impacting growth patterns and antibiotic resistance. This is the first demonstration of a phase-variable Type III DNA methyltransferase in a Gram-positive organism that controls a phasevarion. Characterizing the phenotypic effects of phasevarions in S. suis is key to understanding pathogenesis and the development of future vaccines.IMPORTANCEStreptococcus suis is a causative agent of meningitis, polyarthritis, and polyserositis in swine, and it is a major cause of zoonotic meningitis in humans. Here, we investigate epigenetic gene regulation in S. suis by multiple phasevarions controlled by the phase-variable Type III DNA methyltransferase ModS. This is the first characterized example of a Type III R-M system regulating a phasevarion in a Gram-positive organism. We demonstrate that biphasic ON-OFF switching of ModS expression results in differences in bacterial growth and antibiotic resistance. Understanding the effects of ModS phase variation is required to determine the stably expressed antigenic repertoire of S. suis, which will direct and inform the development of antimicrobial treatments and vaccines against this important pathogen.

Bridging the Gap: A Role for Campylobacter jejuni Biofilms.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis in the developed world. Cases of Campylobacteriosis are common, as the organism is an avian commensal and is passed on to humans through contaminated poultry meat, water, and food preparation areas. Although typically a fastidious organism, C. jejuni can survive outside the avian intestinal tract until it is able to reach a human host. It has long been considered that biofilms play a key role in transmission of this pathogen. The aim of this review is to examine factors that trigger biofilm formation in C. jejuni. A range of environmental elements have been shown to initiate biofilm formation, which are then affected by a suite of intrinsic factors. We also aim to further investigate the role that biofilms may play in the life cycle of this organism.

Assigning a role for chemosensory signal transduction in Campylobacter jejuni biofilms using a combined omics approach.

Biofilms of the gastroenteric pathogen C. jejuni may serve an important role in the transmission of infection from reservoirs of infection to humans. Herein, we undertook a combinatorial approach examining differential gene expression and protein abundance during biofilm formation in C. jejuni. Biofilms induced a substantial rearrangement of the C. jejuni transcriptome and proteome, with ~600 genes differentially expressed when compared to planktonic cells. Genes and proteins induced in biofilms were involved in iron metabolism and acquisition, cell division, glycan production and attachment, while those repressed were associated with metabolism, amino acid usage, and large tracts of the chemotaxis pathway. We further examined the role of chemotaxis in C. jejuni biofilm formation by examining isogenic strains with deletions of the cheV and cheW signal transduction genes. Both ∆cheV and ∆cheW exhibited a significant decrease in directed motility when compared to wild-type C. jejuni as well as demonstrating an increase in autoagglutination ability and biofilm formation. A subtle difference was also observed between the phenotypes of ∆cheV and ∆cheW mutants, both in motility and biofilm formation. This suggests roles for CheV and CheW and may present signal transduction as a potential method for modulating C. jejuni biofilm formation.

RNA Sequencing Data Sets Identifying Differentially Expressed Transcripts during Campylobacter jejuni Biofilm Formation.

Campylobacter jejuni is a foodborne pathogen and an important contributor to gastroenteritis in humans. C. jejuni readily forms biofilms which may play a role in the transmission of the pathogen from animals to humans. Herein, we present RNA sequencing data investigating differential gene expression in biofilm and planktonic C. jejuni These data provide insight into pathways which may be important to biofilm formation in this organism.

Closed Complete Annotated Genome Sequences of Five Haemophilus influenzae Biogroup aegyptius Strains.

Haemophilus influenzae biogroup aegyptius is a cause of conjunctivitis in children. Biogroup aegyptius strains also caused fatal outbreaks of invasive disease, known as Brazilian purpuric fever (BPF), in the 1980s. BPF is fatal if untreated. Here, we report the complete genome sequences of five strains of Haemophilus influenzae biogroup aegyptius.

A direct-sensing galactose chemoreceptor recently evolved in invasive strains of Campylobacter jejuni.

A rare chemotaxis receptor, Tlp11, has been previously identified in invasive strains of Campylobacter jejuni, the most prevalent cause of bacterial gastroenteritis worldwide. Here we use glycan and small-molecule arrays, as well as surface plasmon resonance, to show that Tlp11 specifically interacts with galactose. Tlp11 is required for the chemotactic response of C. jejuni to galactose, as shown using wild type, allelic inactivation and addition mutants. The inactivated mutant displays reduced virulence in vivo, in a model of chicken colonization. The Tlp11 sensory domain represents the first known sugar-binding dCache_1 domain, which is the most abundant family of extracellular sensors in bacteria. The Tlp11 signalling domain interacts with the chemotaxis scaffolding proteins CheV and CheW, and comparative genomic analysis indicates a likely recent evolutionary origin for Tlp11. We propose to rename Tlp11 as CcrG, Campylobacter ChemoReceptor for Galactose.