Analysis of the streptococcal cpsa protein in dna-binding and regulation of capsule and cell wall maintenance

The systemic pathogens Streptococcus agalactiae (GBS) and Streptococcus pneumoniae remain a significant threat to human health worldwide. The ability of these organisms to cause systemic disease is compounded by the production of a polysaccharide capsule that provides immune evasion function. The production of the polysaccharide capsule in systemic streptococcal pathogens is controlled in part by the membrane bound protein CpsA. These studies analyze the contribution of CpsA to regulation of capsule level in the model aquatic pathogen Streptococcus iniae and human specific pathogen GBS, and how this regulation affects virulence in in-vitro, ex-vivo, and in-vivo models of pathogenesis. We have determined that the membrane topology of the CpsA protein consists of a small cytoplasmic N-terminus, and a large extracellular C-terminus that contains the conserved DNA_PPF and LytR protein domains. The cytoplasmic N-terminal region in its entirety is capable of binding specifically to the capsule operon cpsA promoter in S. iniae, and to two putative promoter elements in GBS which include the capsule operon cpsA promoter and the internal cpsE promoter. Additionally, CpsA is a modular protein, with the cytoplasmic N-terminus as a capsule-activating domain, the DNA_PPF region as a capsule-repressing domain, and the LytR region as a control domain that regulates the activities of the other two domains. CpsA also appears to regulate cell wall maintenance, as truncation or removal of CpsA results in longer chains of cocci in both S. iniae and S. agalactiae, a phenotype that is associated with altered antimicrobial resistance and autolysis activity in S. iniae. Taken together, CpsA contributes to the complex regulatory scheme controlling capsule and cell wall, the two major constituents of bacterial cell surface macromolecular structure, and does so in a way that influences pathogenesis during systemic disease. The insights gained through these studies indicates that CpsA can be targeted in a way that is detrimental to bacterial survival in the context of systemic disease, suggesting CpsA may be an important future target of anti-virulence antimicrobial therapy

Identification and characterization of novel virulence factors from the swine pathogen and zoonotic agent streptococcus suis

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Phenotypic and genotypic characterisation of biofilm formation in staphylococcus capitis

Biofilm formation is a major concern in infection caused by S. capitis in newborn babies. Phenotypic and genotypic characteristics of biofilm formation in clinical S. capitis isolates from newborns in Royal Woman’s Hospital in Melbourne were investigated. Of 60 S. capitis isolates, 52 belonged to the subspecies urealyticus and the remaining eight to the subsp. capitis. The most prevalent types, that persisted for 6 years in the unit belonged to subsp. urealyticus. Biofilm formation and antibiotic resistance were also mainly displayed by subsp. urealyticus. Sequence analysis showed that the ica operon in S. capitis was 4160 bp in length. PCR demonstrated its presence in all the isolates. The ica operon harboured in S. capitis is closely related to that of other species of staphylococci. It contains four structural genes icaA, icaD, icaB and icaC, which are co-transcribed from the icaA promoter. A negative regulator icaR gene is located upstream of icaA and transcribed in an opposite orientation. In sillico analysis of ica operon demonstrated the putative function and mechanisms of ICA proteins in polysaccharide biosynthesis and the origin on ica genes present in S. capitis genomes. In vitro biofilm formation and the expression of biofilm-related genes induced by sub-inhibitory concentrations of erythromycin revealed different responses. The expression of icaA and sarA genes was up-regulated, and icaR was down-regulated in the subsp. urealyticus biofilm producer. In contrast, a reverse expression was displayed by the biofilm-negative subsp. capitis isolate. This suggests biofilm formation in S. capitis is regulated by icaR and sarA, and the two subspecies may adopt different regulatory pathways in the two subspecies. The erythromycin induced biofilm formation may have clinical implications according to the doses of erythromycin used clinically. Gene transformation protocols for S. capitis were developed and optimised and will be used as a platform for future work. In summary, this study revealed distinctive phenotypic and genotypic characteristics of the two subspecies of S. capitis which relate to their epidemiology under the clinical setting of newborn intensive care. Isolates of the predominant subsp. urealyticus clones were characterised by their higher antibiotic resistance and biofilm formation ability, and distinct gene expression profiles. Differentiating the subspecies S. capitis revealed will be valuable in understanding their role in bloodstream infections of newborns in hospitals

Understanding the role of different strain types of Fusobacterium necrophorum: biofilms, glycans and metabolic pathways

Fusobacterium necrophorum an obligate Gram-negative anaerobe has been implicated in the cause of persistent severe throat infections and the systemic life-threatening Lemierre’s syndrome; a potentially fatal periodontal disease, which results in abscess formation in the tonsils. The use of antibiotics had led to decreased incidence of F. necrophorum infections to a point that the bacterium became a forgotten pathogen; however, there has recently been a rise in interest. F. necrophorum is thought to survive the aerobic oropharynx by biofilm formation. Studies of optimal conditions for biofilm formation could be useful in improving therapeutic options. This current study determined that strains ARU 01 and JCM 3718 formed the most biofilm at 37 °C, with reduction in biofilm observed at 26 °C and 42 °C. Strain JCM 3724 on the other hand, formed most biofilm at 26 °C and 42 °C; this is an indication that strain JCM 3724 but not JCM 3718 or ARU 01 was able to survive in extreme temperatures by forming biofilms; all strains produced more biofilm at pH 4. Biofilm formation was observed in both mono and dual species culture of F. necrophorum, in dual culture the organisms became resistant to penicillin and ciprofloxacin. As glycans are implicated in biofilm formation, bacterial adhesion to host cells and pathogenicity, the cell surface glycans and cell extracts of F. necrophorum were investigated using enzyme-linked lectin assays (ELLA) and lectin histochemical staining. No significant differences were seen in the staining patterns, but a patchy and variable staining was noted for Sambucus nigra that detects sialic acid. A surface lectin, the Galactose binding protein was identified and characterised as binding to unsubstituted beta galactosyl residues of the type carried by many bacteria suggesting a role in biofilm formation. Subsequent molecular and bioinformatic studies identified all but one key component of the lipid A pathway; lpxI was shown to substitute for lpxH in the pathway. The component genes required for expression of sialic acid on the cell surface of the organism were determined; a polymorphism, the presence or absence of siaA, suggested some but not all strains had the ability to express this sugar on the cell surface. Further studies are required to determine whether this is linked to pathogenicity. Genomic and proteomic studies on type strains and clinical isolates revealed significant differences between subsp. necrophorum and funduliforme that will be useful in developing a simple molecular based subspeciation test. The subsp. funduliforme was split into 3 clusters (A, B and C) based on the genomic data; proteomic studies were used to determine the impact of the non-synonymous SNPs seen; two clusters were observed at the protein level, A and B+C. Most of the amino acid replacements that differentiated the clusters A from B +C were conservative or semi- conservative; more differences were noted between the two subspecies and these also included non-conservative changes that could affect protein structure and function. Clearly, there is scope for further work to elucidate the evolution of these clusters and their relevance to pathogenicity

Characterization of Lactobacillus pili and the niche-adaptation factors of intestinal Lactobacillus ruminis

The mammalian gut microbiota is composed of autochthonous species that permanently colonize the host intestine, and of allochthonous species that are only transiently able to occupy the intestinal environment. In this thesis research, Lactobacillus ruminis and Lactobacillus rhamnosus GG were investigated as paradigms for each type of microbe–host interaction, with special emphasis on the in vitro characterization of their adaptation factors in the host GIT. L. rhamnosus GG has two pilus operons: spaCBA encoding the well-studied SpaCBA pili and spaFED putatively encoding SpaFED pili. The expression of SpaFED pili in L. rhamnosus GG under laboratory conditions has not thus far been reported. In this study, a nisin-induced expression system was used for the generation of SpaFED or SpaF-deleted pili in Lactococcus lactis NZ9000. The results revealed that SpaFED pili were essential in mediating lactococcal adhesion to intestinal cell lines, to certain extracellular matrix proteins, and to porcine mucins, the tip pilin SpaF playing a central role as a focal adhesion factor. With regard to immunomodulation, SpaFED pili appeared to dampen the immune responses, which was largely attributed to the SpaF pilin, in HEK 293-blue cells expressing TLR2, and in Caco-2 cells. While encountering human peripheral blood monocyte-derived dendritic cells, neither immune response enhancement nor immune dampening by SpaFED pili was observed. Consistent with genomic analyses, transmission electron microscopy demonstrated that pili in gut autochthonous L. ruminis ATCC 25644 of human origin consisted of the tip (LrpC), basal (LrpB), and backbone (LrpA) pilins. Recombinant L. lactis strains were constructed, producing either LrpCBA or LrpC-lacking pili of L. ruminis. Recombinant LrpCBA pili mediated lactococcal adherence to ECM proteins and intestinal epithelial cells, and also dampened TLR2-dependent NF-κB signaling and IL-8 production in HEK cells, whereas L. ruminis itself induced evidently elevated immune responses. When incubated with Caco-2 cells, L. ruminis and recombinant lactococcal constructs expressing pili with and without LrpC pilin lowered IL-8 production. Subsequently, a novel L. ruminis strain was isolated from porcine feces and demonstrated to be flagellated and piliated. We analyzed the abilities of this new isolate and three other L. ruminis strains to adhere to host cells and extracellular matrix proteins, to inhibit pathogen binding and growth, to maintain epithelial barrier functions, and to modulate immune responses in vitro. The results indicated that the strains shared several characteristics, such as binding to ECM proteins and HT-29 cells, inhibition of pathogen adhesion and growth, maintenance of epithelial barrier functions in epithelial cells, and activation of innate immune responses to various extents. In conclusion, this thesis study demonstrated the adhesiveness of SpaFED and LrpCBA pili, which may respectively promote the gut retention of L. rhamnosus GG and of L. ruminis. Moreover, pilus-mediated dampening of innate immune responses might be a strategy for these two gut bacterial species to induce immune tolerance in the host. Additionally, L. ruminis inhibited enteropathogen adhesion and growth, as well as maintained intestinal barrier function, which could be regarded as beneficial to the host and which may in turn favor the persistent residence of L. ruminis.not availabl

Odyssée au fil des interfaces: de la physico-chimie des macromolécules à l'enveloppe bactérienne, plate-forme interactive du micro-organisme avec son micro-environnement

This find is registered at Portable Antiquities of the Netherlands with number PAN-0001909