The hijackers guide to escaping complement: Lessons learned from pathogens

Pathogens that invade the human host are confronted by a multitude of defence mechanisms aimed at preventing colonization, dissemination and proliferation. The most frequent outcome of this interaction is microbial elimination, in which the complement system plays a major role. Complement, an essential feature of the innate immune machinery, rapidly identifies and marks pathogens for efficient removal. Consequently, this creates a selective pressure for microbes to evolve strategies to combat complement, permitting host colonization and access to resources. All successful pathogens have developed mechanisms to resist complement activity which are intimately aligned with their capacity to cause disease. In this review, we describe the successful methods various pathogens use to evade complement activation, shut down inflammatory signalling through complement, circumvent opsonisation and override terminal pathway lysis. This review summarizes how pathogens undermine innate immunity: ‘The Hijackers Guide to Complement’

Cytolytic toxin production by \u3ci\u3eStaphylococcus aureus\u3c/i\u3e is dependent upon the activity of the protoheme IX farnesyltransferase

Staphylococcus aureus is a medically important pathogen with an abundance of virulence factors that are necessary for survival within a host, including the production of cytolytic toxins. The regulation of toxin production is mediated by the Agr quorum sensing system, and a poorly defined post-exponential growth phase signal independent of Agr. As part of a recent genome wide association study (GWAS) to identify novel loci that alter the expression of cytolytic toxins, a polymorphism in the cyoE gene, which encodes a protoheme IX farnesyltransferase, was identified. This enzyme is essential for processing heme into the electron transport chain for use as an electron acceptor. Interestingly, without this enzyme S. aureus were repressed in their ability to secrete cytolytic toxins, and this appears to be mediated through repression of the Agr quorum sensing system. We hypothesize that the loss of electron transport is inducing feedback inhibition of metabolic capabilities that suppress the TCA cycle, and that this coupled with decreased RNAIII transcription prevents synthesis of cytolytic toxins

Cigarette smoke exposure redirects Staphylococcus aureus to a virulence profile associated with persistent infection

Altres ajuts: This work also received a grant from the Spanish Society of Pneumology and Thoracic Surgery (SEPAR 024/2016). M.L. gratefully acknowledges funding from the European Respiratory Society Long-term Fellowship grant (LTRF-5934). A.M.E. acknowledges funding from the Royal Society, Department of Medicine (Imperial College), and from the Imperial NIHR Biomedical Research Centre, Imperial College London. B.C.Y is funded by an NIHR Clinical Lectureship. CP acknowledges Programa Germans Trias Sapiens Fundació Catalunya la Pedrera and European Respiratory Society short term research fellowship October 2018 (STRTF201810-00467).Tobacco smoking represents the leading preventable cause of death worldwide. Smoking is a recognised risk factor for several pathologies and is detrimental to host immune surveillance and defence. However, the impact of smoking on microbial residents of the nasopharyngeal cavity, in contact with cigarette smoke (CS), is lacking. Staphylococcus aureus is a major human pathogen that colonises the human nasopharynx and causes a wide range of infections. We investigated the impact of CS on specific virulence phenotypes important in S aureus pathogenesis. We observed strain-dependent differences following exposure to CS, namely growth inhibition, augmented biofilm formation, increased invasion of, and persistence within, bronchial alveolar epithelial cells. Additionally, we confirm the critical role of a functional accessory gene regulator (Agr) system in mediating increased biofilm development and host cell invasion and persistence following CS exposure. Furthermore, CS exposure resulted in reduced toxin production. Importantly, exposure of S aureus to CS accelerated the frequency of mutations and resulted in a significant increase in gentamicin-resistant small colony variant (SCV) formation. Mutational analysis revealed that CS induced SCVs emerge via the SOS response DNA mutagenic repair system. Taken together, our results suggest that CS redirects certain S aureus strains to a virulence profile associated with persistence

Antibacterial Fusion Proteins Enhance Moraxella catarrhalis Killing

Moraxella catarrhalis is a human-specific commensal of the respiratory tract and an opportunistic pathogen. It is one of the leading cause of otitis media in children and of acute exacerbations in patients with chronic obstructive pulmonary disease, resulting in significant morbidity and economic burden. Vaccines and new immunotherapeutic strategies to treat this emerging pathogen are needed. Complement is a key component of innate immunity that mediates the detection, response, and subsequent elimination of invading pathogens. Many pathogens including M. catarrhalis have evolved complement evasion mechanisms, which include the binding of human complement inhibitors such as C4b-binding protein (C4BP) and Factor H (FH). Inhibiting C4BP and FH acquisition by M. catarrhalis may provide a novel therapeutic avenue to treat infections. To achieve this, we created two chimeric proteins that combined the Moraxella-binding domains of C4BP and FH fused to human immunoglobulin Fcs: C4BP domains 1 and 2 and FH domains 6 and 7 fused to IgM and IgG Fc, respectively. As expected, FH6-7/IgG displaced FH from the bacterial surface while simultaneously activating complement via Fc-C1q interactions, together increasing pathogen elimination. C4BP1-2/IgM also increased serum killing of the bacteria through enhanced complement deposition, but did not displace C4BP from the surface of M. catarrhalis. These Fc fusion proteins could act as anti-infective immunotherapies. Many microbes bind the complement inhibitors C4BP and FH through the same domains as M. catarrhalis, therefore these Fc fusion proteins may be promising candidates as adjunctive therapy against many different drug-resistant pathogens

Staphylococcus aureus Interaction with Phospholipid Vesicles – A New Method to Accurately Determine Accessory Gene Regulator (agr) Activity

The staphylococcal accessory gene regulatory (agr) operon is a well-characterised global regulatory element that is important in the control of virulence gene expression for Staphylococcus aureus, a major human pathogen. Hence, accurate and sensitive measurement of Agr activity is central in understanding the virulence potential of Staphylococcus aureus, especially in the context of Agr dysfunction, which has been linked with persistent bacteraemia and reduced susceptibility to glycopeptide antibiotics. Agr function is typically measured using a synergistic haemolysis CAMP assay, which is believe to report on the level of expression of one of the translated products of the agr locus, delta toxin. In this study we develop a vesicle lysis test (VLT) that is specific to small amphipathic peptides, most notably delta and Phenol Soluble Modulin (PSM) toxins. To determine the accuracy of this VLT method in assaying Agr activity, we compared it to the CAMP assay using 89 clinical Staphylococcus aureus isolates. Of the 89 isolates, 16 were designated as having dysfunctional Agr systems by the CAMP assay, whereas only three were designated as such by VLT. Molecular analysis demonstrated that of these 16 isolates, the 13 designated as having a functional Agr system by VLT transcribed rnaIII and secreted delta toxin, demonstrating they have a functional Agr system despite the results of the CAMP assay. The agr locus of all 16 isolates was sequenced, and only the 3 designated as having a dysfunctional Agr system contained mutations, explaining their Agr dysfunction. Given the potentially important link between Agr dysfunction and clinical outcome, we have developed an assay that determines this more accurately than the conventional CAMP assay

Short Leucine-Rich Proteoglycans Modulate Complement Activity and Increase Killing of the Respiratory Pathogen Moraxella catarrhalis

The respiratory pathogen Moraxella catarrhalis is a human-specific commensal that frequently causes acute otitis media in children and stimulates acute exacerbations in chronic obstructive pulmonary disease patients. The exact molecular mechanisms defining host-pathogen interactions promoting pathogenesis are not clearly understood. Limited knowledge hampers vaccine and immunotherapeutic development required to treat this emerging pathogen. In this study, we reveal in detail a novel antibacterial role displayed by short leucine-rich proteoglycans (SLRPs) in concert with complement. We show that fibromodulin (FMOD), osteoadherin (OSAD), and biglycan (BGN) but not decorin (DCN) enhance serum killing of M. catarrhalis. Our results suggest that M. catarrhalis binding to SLRPs is a conserved feature, as the overwhelming majority of clinical and laboratory strains bound all four SLRPs. Furthermore, we resolve the binding mechanism responsible for this interaction and highlight the role of the ubiquitous surface protein (Usp) A2/A2H in mediating binding to host SLRPs. A conserved immune evasive strategy used by M. catarrhalis and other pathogens is the surface acquisition of host complement inhibitors such as C4b-binding protein (C4BP). We observed that FMOD, OSAD, and BGN competitively inhibit binding of C4BP to the surface of M. catarrhalis, resulting in increased C3b/iC3b deposition, membrane attack complex (MAC) formation, and subsequently decreased bacterial survival. Furthermore, both OSAD and BGN promote enhanced neutrophil killing in vitro, both in a complement-dependent and independent fashion. In summary, our results illustrate that SLRPs, FMOD, OSAD, and BGN portray complement-modulating activity enhancing M. catarrhalis killing, defining a new antibacterial role supplied by SLRPs

Significant variability exists in the cytotoxicity of global methicillin-resistant Staphylococcus aureus lineages.

Staphylococcus aureus is a major human pathogen where the emergence of antibiotic resistant lineages, such as methicillin-resistant S. aureus (MRSA), is a major health concern. While some MRSA lineages are restricted to the healthcare setting, the epidemiology of MRSA is changing globally, with the rise of specific lineages causing disease in healthy people in the community. In the past two decades, community-associated MRSA (CA-MRSA) has emerged as a clinically important and virulent pathogen associated with serious skin and soft-tissue infections (SSTI). These infections are primarily cytotoxin driven, leading to the suggestion that hypervirulent lineages/multi-locus sequence types (STs) exist. To examine this, we compared the cytotoxicity of 475 MRSA isolates representing five major MRSA STs (ST22, ST93, ST8, ST239 and ST36) by employing a monocyte-macrophage THP-1 cell line as a surrogate for measuring gross cytotoxicity. We demonstrate that while certain MRSA STs contain highly toxic isolates, there is such variability within lineages to suggest that this aspect of virulence should not be inferred from the genotype of any given isolate. Furthermore, by interrogating the accessory gene regulator (Agr) sequences in this collection we identified several Agr mutations that were associated with reduced cytotoxicity. Interestingly, the majority of isolates that were attenuated in cytotoxin production contained no mutations in the agr locus, indicating a role of other undefined genes in S. aureus toxin regulation

Molecular analysis of accessory gene regulator functionality and virulence genes in Staphylococcus aureus derived from pediatric wound infections

Staphylococcus aureus is a major human pathogen causing infections with high morbidity and mortality in both healthcare and community settings. The accessory gene regulator (Agr) is a key genetic element controlling the expression of numerous virulence factors in S. aureus. The significance of a functional Agr system in clinical S. aureus isolates derived from pediatric wound infections is still unclear. Therefore, the present study was conducted to identify virulence genes and determine Agr functionality from this cohort of patients. A total of 48 S. aureus wound isolates were collected from patients referred to Tehran Children's Medical Center Hospital from April 2017 to April 2018. In addition, in vitro antimicrobial susceptibility of the isolates was assessed using the disk diffusion and E-test methods. Conventional PCR was performed for the detection of toxins (tsst-1, hla, hlb, hld, eta, etb, etd, edin-A, edin-B, edin-C) and Agr typing (agrI, agrII, agrIII, agrIV). Agr functionality was assessed by quantitative reverse transcriptase real-time PCR (qRT-PCR). All S. aureus isolates were found to be susceptible to linezolid and vancomycin. The most frequently detected toxin gene was eta (100%), and the most prevalent Agr type was agrIII (56.3%). Importantly, qRT-PCR revealed that Agr was functional in 28 (58%) of wound isolates. Consequently, our data suggests that a functional Agr system may not be required for the development of S. aureus wound infections.</p

A new assay for rhamnolipid detection-important virulence factors of <em>Pseudomonas aeruginosa</em>

Rhamnolipids (RLs) are heterogeneous glycolipid molecules that are composed of one or two L-rhamnose sugars and one or two β-hydroxy fatty acids, which can vary in their length and branch size. They are biosurfactants, predominantly produced by Pseudomonas aeruginosa and are important virulence factors, playing a major role in P. aeruginosa pathogenesis. Therefore, a fast, accurate and high-throughput method of detecting such molecules is of real importance. Here, we illustrate the ability to detect RL-producing P. aeruginosa strains with high sensitivity, based on an assay involving phospholipid vesicles encapsulated with a fluorescent dye. This vesicle-lysis assay is confirmed to be solely sensitive to RLs. We illustrate a half maximum concentration for vesicle lysis (EC50) of 40 μM (23.2 μg/mL) using pure commercial RLs and highlight the ability to semi-quantify RLs directly from the culture supernatant, requiring no extra extraction or processing steps or technical expertise. We show that this method is consistent with results from thin-layer chromatography detection and dry weight analysis of RLs but find that the widely used orcinol colorimetric test significantly underestimated RL quantity. Finally, we apply this methodology to compare RL production among strains isolated from either chronic or acute infections. We confirm a positive association between RL production and acute infection isolates (p = 0.0008), highlighting the role of RLs in certain infections.</p