Absence of protein A expression is associated with higher capsule production in staphylococcal isolates

Staphylococcus aureus is a major human pathogen, and a leading cause of soft tissue and blood stream infections. One of the causes of its success as a pathogen is the peculiar array of immune evasion factors through which the bacterium avoids host defenses, where the staphylococcal protein A (SpA) plays a major role thanks to its IgG binding activities. Moreover, SpA has recently been proposed as a promising vaccine antigen. In this study, we evaluated the expression of SpA in a collection of staphylococcal strains, about 7% of which did not express SpA (SpA- strains), despite the presence of the gene. By a comparative genomic analysis, we identified that a mutation in the spa 5′ UTR sequence affecting the RBS is responsible for the loss of SpA in a subset of SpA- strains. Using a high-throughput qRT-PCR approach on a selected panel of virulence-related genes, we identified that the SpA- phenotype is associated with lower spa transcript levels and increased expression and production of capsule as well as other changes in the transcription of several key virulence factors. Our data suggest that the SpA- phenotype has occurred in geographically distinct strains through different molecular mechanisms including both mutation, leading likely to translation alterations, and transcriptional deregulation. Furthermore, we provide evidence that SpA- strains are highly susceptible to phagocytic uptake mediated by anti-capsule antibodies. These data suggest that S. aureus may alter its virulence factor expression pattern as an adaptation to the host or environment. Vaccination strategies targeting both SpA and capsule could therefore result in broader coverage against staphylococcal isolates than SpA alone

Staphylococcus aureus in animals

Staphylococcus aureus is a mammalian commensal and opportunistic pathogen that colonizes niches such as skin, nares and diverse mucosal membranes of about 20-30% of the human population. S. aureus can cause a wide spectrum of diseases in humans and both methicillin-sensitive and methicillin-resistant strains are common causes of nosocomial- and community-acquired infections. Despite the prevalence of literature characterising staphylococcal pathogenesis in humans, S. aureus is a major cause of infection and disease in a plethora of animal hosts leading to a significant impact on public health and agriculture. Infections in animals are deleterious to animal health, and animals can act as a reservoir for staphylococcal transmission to humans. Host-switching events between humans and animals and amongst animals are frequent and have been accentuated with the domestication and/or commercialisation of specific animal species. Host-switching is typically followed by subsequent adaptation through acquisition and/or loss of mobile genetic elements such as phages, pathogenicity islands and plasmids as well as further host-specific mutations allowing it to expand into new host populations. In this chapter, we will be giving an overview of S. aureus in animals, how this bacterial species was, and is, being transferred to new host species and the key elements thought to be involved in its adaptation to new ecological host niches. We will also highlight animal hosts as a reservoir for the development and transfer of antimicrobial resistance determinants

In vivo analysis of staphylococcus aureus-infected mice reveals differential temporal and spatial expression patterns of fhuD2

Staphylococcus aureus is an opportunistic human pathogen and a major cause of invasive infections such as bacteremia, endocarditis, pneumonia and wound infections. FhuD2 is a staphylococcal lipoprotein involved in the uptake of iron-hydroxymate and is under the control of the iron uptake regulator Fur. The protein is part of an investigational multi-component vaccine formulation that has shown protective efficacy in several murine models of infection. Even though fhuD2 expression was shown to be upregulated in murine kidneys infected with S. aureus, it is unknown whether the bacterium undergoes increased iron deprivation during prolonged infection. Furthermore, different infection niches of S. aureus might provide different environments and iron availability resulting in different fhuD2 expression pattern within different host organs. To address these questions, we characterized the in vitro expression of the fhuD2 gene and confirmed Fur-dependent iron-regulation of its expression. We further investigated its expression in mice infected with a bioluminescent reporter strain of S. aureus expressing the luciferase operon under the control of the fhuD2 promoter. The emission of bioluminescence in different organs was followed over a seven-day time course, as well as quantitative real-time PCR analysis of the RNA transcribed from the endogenous fhuD2 gene. Using this approach, we could show that fhuD2 expression was induced during infection in all organs analyzed and that differences in expression were observed in the temporal expression profiles, and between infected organs. Our data suggest that S. aureus undergoes increased iron deprivation during progression of infection in diverse host organs and accordingly induces dedicated iron acquisition mechanisms. Since FhuD2 plays a central role in providing the pathogen with the required iron, further knowledge of the patterns of fhuD2 expression in vivo during infection is instrumental in better defining the role of this antigen in S. aureus pathogenesis and as a vaccine antigen

Importance of Lipopolysaccharide and Cyclic β-1,2-Glucans in Brucella-Mammalian Infections

Brucella species are the causative agents of one of the most prevalent zoonotic diseases: brucellosis. Infections by Brucella species cause major economic losses in agriculture, leading to abortions in infected animals and resulting in a severe, although rarely lethal, debilitating disease in humans. Brucella species persist as intracellular pathogens that manage to effectively evade recognition by the host's immune system. Sugar-modified components in the Brucella cell envelope play an important role in their host interaction. Brucella lipopolysaccharide (LPS), unlike Escherichia coli LPS, does not trigger the host's innate immune system. Brucella produces cyclic β-1,2-glucans, which are important for targeting them to their replicative niche in the endoplasmic reticulum within the host cell. This paper will focus on the role of LPS and cyclic β-1,2-glucans in Brucella-mammalian infections and discuss the use of mutants, within the biosynthesis pathway of these cell envelope structures, in vaccine development

Phage-inducible chromosomal islands promote genetic variability by blocking phage reproduction and protecting transductants from phage lysis

Funding: This work was supported by the following grants awarded to JPR: MR/M003876/1, MR/S00940X/1 and MR/V000772/1 from the Medical Research Council (MRC, UK; https://mrc.ukri.org); BB/N002873/1, BB/S003835/1 and BB/V002376/1 from the Biotechnology and Biological Sciences Research Council (BBSRC, UK; https://bbsrc.ukri.org); 201531/Z/16/Z from the Wellcome Trust (https://wellcome.org).Phage-inducible chromosomal islands (PICIs) are a widespread family of highly mobile genetic elements that disseminate virulence and toxin genes among bacterial populations. Since their life cycle involves induction by helper phages, they are important players in phage evolution and ecology. PICIs can interfere with the lifecycle of their helper phages at different stages resulting frequently in reduced phage production after infection of a PICI-containing strain. Since phage defense systems have been recently shown to be beneficial for the acquisition of exogenous DNA via horizontal gene transfer, we hypothesized that PICIs could provide a similar benefit to their hosts and tested the impact of PICIs in recipient strains on host cell viability, phage propagation and transfer of genetic material. Here we report an important role for PICIs in bacterial evolution by promoting the survival of phage-mediated transductants of chromosomal or plasmid DNA. The presence of PICIs generates favorable conditions for population diversification and the inheritance of genetic material being transferred, such as antibiotic resistance and virulence genes. Our results show that by interfering with phage reproduction, PICIs can protect the bacterial population from phage attack, increasing the overall survival of the bacterial population as well as the transduced cells. Moreover, our results also demonstrate that PICIs reduce the frequency of lysogenization after temperate phage infection, creating a more genetically diverse bacterial population with increased bet-hedging opportunities to adapt to new niches. In summary, our results identify a new role for the PICIs and highlight them as important drivers of bacterial evolution.Publisher PDFPeer reviewe

Multilayer regulation of Neisseria meningitidis NHBA at physiologically relevant temperatures

This research was sponsored by GlaxoSmithKline Biologicals SA. A.F.H. was funded by Marie Sklodowska-Curie Intra-European Fellowships (PIEF-GA-2012-328377).Neisseria meningitidis colonizes the nasopharynx of humans, and pathogenic strains can disseminate into the bloodstream, causing septicemia and meningitis. NHBA is a surface-exposed lipoprotein expressed by all N. meningitidis strains in different isoforms. Diverse roles have been reported for NHBA in heparin-mediated serum resistance, biofilm formation, and adherence to host tissues. We determined that temperature controls the expression of NHBA in all strains tested, with increased levels at 30–32 °C compared to 37 °C. Higher NHBA expression at lower temperatures was measurable both at mRNA and protein levels, resulting in higher surface exposure. Detailed molecular analysis indicated that multiple molecular mechanisms are responsible for the thermoregulated NHBA expression. The comparison of mRNA steady-state levels and half-lives at 30 °C and 37 °C demonstrated an increased mRNA stability/translatability at lower temperatures. Protein stability was also impacted, resulting in higher NHBA stability at lower temperatures. Ultimately, increased NHBA expression resulted in higher susceptibility to complement-mediated killing. We propose that NHBA regulation in response to temperature downshift might be physiologically relevant during transmission and the initial step(s) of interaction within the host nasopharynx. Together these data describe the importance of NHBA both as a virulence factor and as a vaccine antigen during neisserial colonization and invasion.Publisher PDFPeer reviewe

Multivalent grafting of hyperbranched oligo- and polyglycerols shielding rough membranes to mediate hemocompatibility

Hemocompatible materials are needed for internal and extracorporeal biomedical applications, which should be realizable by reducing protein and thrombocyte adhesion to such materials. Polyethers have been demonstrated to be highly efficient in this respect on smooth surfaces. Here, we investigate the grafting of oligo- and polyglycerols to rough poly(ether imide) membranes as a polymer relevant to biomedical applications and show the reduction of protein and thrombocyte adhesion as well as thrombocyte activation. It could be demonstrated that, by performing surface grafting with oligo- and polyglycerols of relatively high polydispersity (>1.5) and several reactive groups for surface anchoring, full surface shielding can be reached, which leads to reduced protein adsorption of albumin and fibrinogen. In addition, adherent thrombocytes were not activated. This could be clearly shown by immunostaining adherent proteins and analyzing the thrombocyte covered area. The presented work provides an important strategy for the development of application relevant hemocompatible 3D structured materials

The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence

K.L.S. was supported by the Australian National Health and Medical Research Council (NHMRC) C. J. Martin Fellowship and Career Development Fellowship. A.F.H. was supported by a Marie Curie Fellowship (PIEF-GA-2012-328377). F.O., L.F., and S.B. were recipients of Novartis fellowships from the Ph.D. program of the University of Siena (Siena, Italy) and University of Bologna (Bologna, Italy), respectively.GNA2091 is one of the components of the 4-component meningococcal serogroup B vaccine (4CMenB) vaccine and is highly conserved in all meningococcal strains. However, its functional role has not been fully characterized. Here we show that nmb2091 is part of an operon and is cotranscribed with the nmb2089, nmb2090, and nmb2092 adjacent genes, and a similar but reduced operon arrangement is conserved in many other gram-negative bacteria. Deletion of the nmb2091 gene causes an aggregative phenotype with a mild defect in cell separation; differences in the outer membrane composition and phospholipid profile, in particular in the phosphoethanolamine levels; an increased level of outer membrane vesicles; and deregulation of the zinc-responsive genes such as znuD. Finally, the Δ2091 strain is attenuated with respect to the wild-type strain in competitive index experiments in the infant rat model of meningococcal infection. Altogether these data suggest that GNA2091 plays important roles in outer membrane architecture, biogenesis, homeostasis, and in meningococcal survival in vivo, and amodel for its role is discussed. These findings highlight the importance of GNA2091 as a vaccine component.PostprintPeer reviewe

Resummation of mass terms in perturbative massless quantum field theory

The neutral massless scalar quantum field $\Phi$ in four-dimensional space-time is considered, which is subject to a simple bilinear self-interaction. Is is well-known from renormalization theory that adding a term of the form $-\frac{m^2}{2} \Phi^2$ to the Lagrangean has the formal effect of shifting the particle mass from the original zero value to m after resummation of all two-leg insertions in the Feynman graphs appearing in the perturbative expansion of the S-matrix. However, this resummation is accompanied by some subtleties if done in a proper mathematical manner. Although the model seems to be almost trivial, is shows many interesting features which are useful for the understanding of the convergence behavior of perturbation theory in general. Some important facts in connection with the basic principles of quantum field theory and distribution theory are highlighted, and a remark is made on possible generalizations of the distribution spaces used in local quantum field theory. A short discussion how one can view the spontaneous breakdown of gauge symmetry in massive gauge theories within a massless framework is presented.Comment: 15 pages, LaTeX (style files included), one section adde

Gauge-invariant Green's functions for the bosonic sector of the standard model

There are many applications in gauge theories where the usually employed framework involving gauge-dependent Green's functions leads to considerable problems. In order to overcome the difficulties invariably tied to gauge dependence, we present a manifestly gauge-invariant approach. We propose a generating functional of appropriately chosen gauge-invariant Green's functions for the bosonic sector of the standard model. Since the corresponding external sources emit one-particle states, these functions yield the same S-matrix elements as those obtained in the usual framework. We evaluate the generating functional for the bosonic sector of the standard model up to the one-loop level and carry out its renormalization in the on-shell scheme. Explicit results for some two-point functions are given. Gauge invariance is manifest at any step of our calculation.Comment: 29 pages, Revtex. v2: Discussions improved, conclusions unchanged. Some references added. v3: Published versio