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Characterization of a leukocidin identified in Staphylococcus pseudintermedius.

ABSTRACT: Bacterial infections from Staphylococcus pseudintermedius are the most common cause of skin infections (pyoderma) affecting dogs. Two component pore-forming leukocidins are a family of potent toxins secreted by staphylococci and consist of S (slow) and F (fast) components. They impair the innate immune system, the first line of defense against these pathogens. Seven different leukocidins have been characterized in Staphylococcus aureus, some of which are host and cell specific. Through genome sequencing and analysis of the S. pseudintermedius secretome using liquid chromatography mass spectrometry we identified two proteins, named "LukS-I" and "LukF-I", encoded on a degenerate prophage contained in the genome of S. pseudintermedius isolates. Phylogenetic analysis of LukS-I components in comparison to the rest of the leukocidin family showed that LukS-I was most closely related to S. intermedius LukS-I, S. aureus LukE and LukP, whereas LukF-I was most similar to S. intermedius LukF-I S. aureus gamma hemolysin subunit B. The killing effect of recombinant S. pseudintermedius LukS-I and LukF-I on canine polymorphonuclear leukocytes was determined using a flow cytometry cell permeability assay. The cytotoxic effect occurred only when the two recombinant proteins were combined. Engineered mutant versions of the two-component pore-forming leukocidins, produced through amino acids substitutions at selected points, were not cytotoxic. Anti-Luk-I produced in dogs against attenuated proteins reduced the cytotoxic effect of native canine leukotoxin which highlights the importance of Luk-I as a promising component in a vaccine against canine S. pseudintermedius infections.

SUBMITTER: Abouelkhair MA

PROVIDER: S-EPMC6160070 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Characterization of a leukocidin identified in Staphylococcus pseudintermedius.

Abouelkhair Mohamed A MA Bemis David A DA Giannone Richard J RJ Frank Linda A LA Kania Stephen A SA

PloS one 20180927 9

Bacterial infections from Staphylococcus pseudintermedius are the most common cause of skin infections (pyoderma) affecting dogs. Two component pore-forming leukocidins are a family of potent toxins secreted by staphylococci and consist of S (slow) and F (fast) components. They impair the innate immune system, the first line of defense against these pathogens. Seven different leukocidins have been characterized in Staphylococcus aureus, some of which are host and cell specific. Through genome se ...[more]

PMID: 30261001

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Project description:BACKGROUND:Staphylococcus pseudintermedius is an opportunistic pathogen that is the major cause of pyoderma affecting dogs. Conventional antimicrobial treatment for infections caused by this organism have failed in recent years due to widespread resistance and alternative treatment strategies are a high priority. Protein A encoded in Staphylococcus aureus by spa protects the bacterium by binding IgG and acts as a superantigen. Staphylococcus pseudintermedius possess two genes orthologous to S. aureus spa, spsP, and spsQ. METHODS:SpsQ and SpsQ-M, a non-toxigenic SpsQ, were cloned and expressed as recombinant proteins and their cytotoxic effect on canine B cells was measured. The neutralizing ability of antibody raised against them in clinically healthy dogs was evaluated. RESULTS:S. pseudintermedius SpsQ induced apoptosis of canine B cells. Specific amino acid substitutions diminished SpsQ-M binding to immunoglobulin and its super-antigenic activity, while its antigenicity was maintained. This recombinant, non-toxigenic S. pseudintermedius SpsQ stimulated the production of antibodies in dogs that specifically reacted with SpsQ and greatly diminished its cytotoxic effect on canine B cells. CONCLUSIONS:The production of neutralizing antibody suggests that attenuated, non-toxic SpsQ produced in this study is a good candidate for inclusion in a vaccine for use in the treatment and prevention of S. pseudintermedius infections. ABBREVIATIONS:SpA: Staphylococcus aureus protein A; SpsP: Staphylococcus pseudintermedius protein A; SpsQ: Staphylococcus pseudintermedius protein A; SpsQ-M: attenuated Staphylococcus pseudintermedius protein A; MRSP: methicillin resistant Staphylococcus pseudintermedius; IgA: immunoglobulin A; IgG: immunoglobulin G; IgM: immunoglobulin M; VH: variable region of immunoglobulin heavy chain; IgBD: immunoglobulin binding domains; MFI: mean fluorescent intensity; SEM: standard error of the mean; PBMC: Peripheral blood mononuclear cells; CD21: complement receptor type 2; ST: Sequence type; OD: Optical density; ORF: open reading frame; PBS: Phosphate buffered saline; Tween 20: Polyethylene glycol sorbitan monolaurate 20; HRP: horseradish peroxidase; TMB- 3,3',5,5'-Tetramethylbenzidine.

Project description:Staphylococcus pseudintermedius is an important canine pathogen implicated in an increasing number of human infections. Along with rising levels of methicillin and multidrug resistance, staphylococcal biofilms are a complicating factor for treatment and contribute to device, implant, and surgical infections. Staphylococcal virulence, including biofilm formation, is regulated in part by the quorum sensing accessory gene regulator system (agr). The signal molecule for agr, known as the autoinducing peptide molecule, contains polymorphisms that result in the formation of distinct groups. In S. pseudintermedius, 4 groups (i.e., groups I, II, III, and IV) have been identified but not comprehensively examined for associations with infection type, virulence factor carriage, or phylogenetic relationships-all of which have been found to be significant in S. aureus In this study, 160 clinical canine isolates from Texas, including isolates from healthy dogs (n?=?40) and 3 different infection groups (pyoderma, urinary tract, and surgical, n?=?40 each), were sequenced. The agr group, biofilm-producing capabilities, toxin gene carriage, antimicrobial resistance, and sequence type (ST) were identified for all isolates. While no significant associations were discovered among the clinical infection types and agr groups, agr II isolates were significantly less common than any other group in diseased dogs. Furthermore, agr II isolates were less likely than other agr groups to be multidrug resistant and to carry toxin genes expA and sec-canine Fifty-two (33%) of the 160 isolates were methicillin resistant, and the main sequence types (ST64, ST68, ST71, ST84, ST150, and ST155) of methicillin-resistant strains of S. pseudintermedius (MRSP) were identified for the geographic region.IMPORTANCE Staphylococcus pseudintermedius is an important disease-causing bacterium in dogs and is recognized as a growing threat to human health. Due to increasing multidrug resistance, discovery of alternative methods for treatment of these infections is vital. Interference with one target for alternative treatment, the quorum sensing system agr, has demonstrated clinical improvement of infections in S. aureus animal models. In this study, we sequenced and characterized 160 clinical S. pseudintermedius isolates and their agr systems in order to increase understanding of the epidemiology of the agr group and clarify its associations with types of infection and antimicrobial resistance. We found that isolates with agr type II were significantly less common than other agr types in diseased dogs. This provides valuable information to veterinary clinical microbiologists and clinicians, especially as less research has been performed on infection associations of agr and its therapeutic potential in S. pseudintermedius than in S. aureus.

Dataset Information

Characterization of a leukocidin identified in Staphylococcus pseudintermedius.

Publications

Characterization of a leukocidin identified in Staphylococcus pseudintermedius.

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