Surface layer proteins isolated from Clostridium difficile induce clearance responses in macrophages

Collins, Laura; Lynch, Mark; Marszalowska, Izabela; Kristek, Maja; Rochfort, Keith D.; O’Connell, Mary J.; Windle, Henry J.; Kelleher, Dermot; Loscher, Christine E.

doi:10.1016/j.micinf.2014.02.001

Cited by 23 publications

(19 citation statements)

References 36 publications

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“…We have previously shown that RT 001 SLPs activate macrophages and dendritic cells to produce pro-inflammatory cytokines [25, 26] and the profile of cytokines induced was comparable to that of LPS stimulation. In this study we observed that SLPs from different ribotypes elicited distinct responses in macrophages.…”

Section: Discussionmentioning

confidence: 99%

“…Specifically, we showed that SLPs from RT 001 activated TLR4 signalling, inducing the maturation of dendritic cells in vitro and subsequent T helper cell activation [25]. More recently, we have shown that 001 SLPs induce clearance responses in macrophages [26] and other studies have also shown SLPs from RT 001 to effectively induce an immune response [27, 28]. Together these findings provide a mechanism for interaction between host and pathogen.…”

Section: Introductionmentioning

confidence: 91%

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Surface layer proteins from virulent Clostridium difficile ribotypes exhibit signatures of positive selection with consequences for innate immune response

et al. 2017

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Background Clostridium difficile is a nosocomial pathogen prevalent in hospitals worldwide and increasingly common in the community. Sequence differences have been shown to be present in the Surface Layer Proteins (SLPs) from different C. difficile ribotypes (RT) however whether these differences influence severity of infection is still not clear.ResultsWe used a molecular evolutionary approach to analyse SLPs from twenty-six C. difficile RTs representing different slpA sequences. We demonstrate that SLPs from RT 027 and 078 exhibit evidence of positive selection (PS). We compared the effect of these SLPs to those purified from RT 001 and 014, which did not exhibit PS, and demonstrate that the presence of sites under positive selection correlates with ability to activate macrophages. SLPs from RTs 027 and 078 induced a more potent response in macrophages, with increased levels of IL-6, IL-12p40, IL-10, MIP-1α, MIP-2 production relative to RT 001 and 014. Furthermore, RTs 027 and 078 induced higher expression of CD40, CD80 and MHC II on macrophages with decreased ability to phagocytose relative to LPS.ConclusionsThese results tightly link sequence differences in C. difficile SLPs to disease susceptibility and severity, and suggest that positively selected sites in the SLPs may play a role in driving the emergence of hyper-virulent strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-017-0937-8) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 91%

Surface layer proteins from virulent Clostridium difficile ribotypes exhibit signatures of positive selection with consequences for innate immune response

et al. 2017

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“…The outer surface of many bacteria is covered in a proteinaceous coat called the S-layer (surface layer) that is involved in growth, function, and interaction with the host (Fagan and Fairweather 2014 ). In Gram-positive species, such as C. difficile , SLPs have been shown to play a role in adherence to gastrointestinal tract cells and extracellular matrix components (Calabi et al 2002 ; Takumi et al 1991 ), and recently, SLPs were shown to have a role in activating innate and adaptive immunity through TLR4 (Ryan et al 2011 ) and induce pro-inflammatory cytokines (Bianco et al 2011 ; Collins et al 2014 ). It has been known for several years that patients with recurrent episodes of C. difficile have significantly lower anti-SLP IgM titers than patients experiencing a single episode of C. difficile infection (Drudy et al 2004 ).…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, C. difficile SLPs play a critical role in bacterial adherence to host cells (Calabi et al 2002 ; Drudy et al 2001 ; Merrigan et al 2013 ; Takumi et al 1991 ) and thereby contribute to colonization and the persistence of infection. They have also been shown to perturb cytokine homeostasis and modulate immune responses (Ausiello et al 2006 ; Bianco et al 2011 ; Collins et al 2014 ; Ryan et al 2011 ). SLPs induce maturation of dendritic cells and the subsequent generation of a T-helper cell response through Toll-like receptor 4 (TLR4), thereby altering host inflammatory and regulatory cytokines toward an inflammatory state and contributing to the damage of the intestinal epithelium.…”

Section: Introductionmentioning

confidence: 99%

Targeting surface-layer proteins with single-domain antibodies: a potential therapeutic approach against Clostridium difficile-associated disease

Kandalaft

Hussack

Aubry

et al. 2015

Appl Microbiol Biotechnol

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Clostridium difficile is a leading cause of death from gastrointestinal infections in North America. Antibiotic therapy is effective, but the high incidence of relapse and the rise in hypervirulent strains warrant the search for novel treatments. Surface layer proteins (SLPs) cover the entire C. difficile bacterial surface, are composed of high-molecular-weight (HMW) and low-molecular-weight (LMW) subunits, and mediate adherence to host cells. Passive and active immunization against SLPs has enhanced hamster survival, suggesting that antibody-mediated neutralization may be an effective therapeutic strategy. Here, we isolated a panel of SLP-specific single-domain antibodies (VHHs) using an immune llama phage display library and SLPs isolated from C. difficile hypervirulent strain QCD-32g58 (027 ribotype) as a target antigen. Binding studies revealed a number of VHHs that bound QCD-32g58 SLPs with high affinity (KD = 3–6 nM) and targeted epitopes located on the LMW subunit of the SLP. The VHHs demonstrated melting temperatures as high as 75 °C, and a few were resistant to the gastrointestinal protease pepsin at physiologically relevant concentrations. In addition, we demonstrated the binding specificity of the VHHs to the major C. difficile ribotypes by whole cell ELISA, where all VHHs were found to bind 001 and 027 ribotypes, and a subset of antibodies were found to be broadly cross-reactive in binding cells representative of 012, 017, 023, and 078 ribotypes. Finally, we showed that several of the VHHs inhibited C. difficile QCD-32g58 motility in vitro. Targeting SLPs with VHHs may be a viable therapeutic approach against C. difficile-associated disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6594-1) contains supplementary material, which is available to authorized users.

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“…Nontoxin virulence factors, such as surface layer proteins (SLPs) and cell wall proteins, have also been described and may play a role in disease expression (7)(8)(9).…”

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confidence: 99%

Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis

Negm

Hamed

Dilnot

et al. 2015

Clin Vaccine Immunol

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g Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotypespecific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. C lostridium difficile is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea, imposing a considerable financial burden on health service providers in both Europe and the United States (1-3). Infection causes a spectrum of clinical presentations ranging from an asymptomatic carrier state to severe fulminant colitis and death (4). Following successful treatment, an estimated 20% to 30% of patients with primary C. difficile infection (CDI) develop recurrence of symptoms, caused by either relapse of the original infection or reinfection with a new strain (5).This anaerobic and spore-forming bacterium exerts its major pathological effects through two proinflammatory and cytotoxic protein exotoxins, TcdA (toxin A) and TcdB (toxin B) (6). Nontoxin virulence factors, such as surface layer proteins (SLPs) and cell wall proteins, have also been described and may play a role in disease expression (7-9).The majority of healthy adults have detectable antibodies to C. difficile TcdA and TcdB in their sera that are thought to arise from colonization in infancy or from repeated exposure to C. difficile in adulthood from the environment (10, 11). Several clinical studies suggest that adaptive humoral immune responses, in particular to TcdA and TcdB, may influence clinical outcomes of CDI (12). Most notably, a landmark study in 2000 reported that a low IgG titer to TcdA, but not TcdB, at the time of infection is associated with development of symptomatic disease (13). More recently, the same group demonstrated an association between median IgG titers to TcdA and 30-day all-cause mortality (14). Several r...

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Surface layer proteins isolated from Clostridium difficile induce clearance responses in macrophages

Cited by 23 publications

References 36 publications

Surface layer proteins from virulent Clostridium difficile ribotypes exhibit signatures of positive selection with consequences for innate immune response

Surface layer proteins from virulent Clostridium difficile ribotypes exhibit signatures of positive selection with consequences for innate immune response

Targeting surface-layer proteins with single-domain antibodies: a potential therapeutic approach against Clostridium difficile-associated disease

Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis

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