Virulence of Staphylococcus aureus in a mouse mastitis model: studies of alpha hemolysin, coagulase, and protein A as possible virulence determinants with protoplast fusion and gene cloning.

ABSTRACT Carbon catabolite protein A (CcpA) is known to function as a major regulator of gene expression in different gram-positive organisms. Deletion of the ccpA homologue (saCOL1786) in Staphylococcus aureus was found to affect growth, glucose metabolization, and transcription of selected virulence determinants. In liquid culture, deletion of CcpA decreased the growth rate and yield; however, the effect was only transient during the exponential-growth phase as long as glucose was present in the medium. Depletion of glucose and production of lactate was delayed, while the level of excretion of acetate was less affected and was even higher in the mutant culture. On solid medium, in contrast, growth of the ΔccpA mutant resulted in smaller colonies containing a lower number of CFU per colony. Deletion of CcpA had an effect on the expression of important virulence factors of S. aureus by down-regulating RNAIII, the effector molecule of the agr locus, and altering the transcription patterns of hla, encoding α-hemolysin, and spa, encoding protein A. CcpA inactivation markedly reduced the oxacillin resistance levels in the highly methicillin-resistant S. aureus strain COLn and the teicoplanin resistance level in a glycopeptide-intermediate-resistant S. aureus strain. The presence of CcpA in the capsular polysaccharide serotype 5 (CP5)-producing strain Newman abolished capsule formation and decreased cap operon transcription in the presence of glucose. The staphylococcal CcpA thus not only is involved in the regulation of carbon metabolism but seems to function as a modulator of virulence gene expression as well.

Download Full-text

Prevalence of Methicillin Resistant and Virulence Determinants in Clinical Isolates of Staphylococcus aureus

The Open Infectious Diseases Journal ◽

10.2174/1874279301810010108 ◽

2018 ◽

Vol 10 (1) ◽

pp. 108-115

Author(s):

Manjunath Chavadi ◽

Rahul Narasanna ◽

Ashajyothi Chavan ◽

Ajay Kumar Oli ◽

Chandrakanth Kelmani. R

Keyword(s):

Staphylococcus Aureus ◽

Cell Morphology ◽

Methicillin Resistance ◽

Protein A ◽

Clinical Isolates ◽

Clinical Samples ◽

Virulence Determinants ◽

Methicillin Resistant ◽

Alternative Medicines ◽

Mrsa Strains

Introduction:Methicillin-resistantStaphylococcus aureus(MRSA) is the major threat that is a result of the uncontrolled use of antibiotics causing a huge loss in health, so understanding their prevalence is necessary as a public health measure.Objective:The aim of this study was to determine the prevalence of methicillin-resistant MRSA and virulence determinant among associatedS. aureusfrom the clinical samples obtained from various hospital and health care centers of the Gulbarga region in India.Materials and Methods:All the collected samples were subjected for the screening ofS. aureusand were further characterized by conventional and molecular methods including their antibiotic profiling. Further, the response of methicillin antibiotic on cell morphology was studied using scanning electron microscopy.Results:A total 126S. aureuswas isolated from the clinical samples which showed, 100% resistant to penicillin, 55.5% to oxacillin, 75.3% to ampicillin, 70.6% to streptomycin, 66.6% to gentamicin, 8.7% to vancomycin and 6.3% to teicoplanin. The selected MRSA strains were found to possessmecA(gene coding for penicillin-binding protein 2A) andfemA(factor essential for methicillin resistance)genetic determinants in their genome with virulence determinants such as Coagulase (coa) and the X region of the protein A (spa)gene. Further, the methicillin response in resistantS. aureusshowed to be enlarged and malformed on cell morphology.Conclusion:The molecular typing of clinical isolates ofS. aureusin this study was highly virulent and also resistant to methicillin; this will assist health professionals to control, exploration of alternative medicines and new approaches to combat Staphylococcal infections more efficiently by using targeted therapy.

Download Full-text

mgr, a Novel Global Regulator in Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.185.13.3703-3710.2003 ◽

2003 ◽

Vol 185 (13) ◽

pp. 3703-3710 ◽

Cited By ~ 107

Author(s):

Thanh T. Luong ◽

Steven W. Newell ◽

Chia Y. Lee

Keyword(s):

Staphylococcus Aureus ◽

Capsular Polysaccharide ◽

Polyacrylamide Gel Electrophoresis ◽

Protein A ◽

Sodium Dodecyl ◽

Binding Motif ◽

Transcriptional Level ◽

Virulence Determinants ◽

Alpha Toxin ◽

Extracellular Protein Production

ABSTRACT The virulence determinants of Staphylococcus aureus are coordinately controlled by several unlinked chromosomal loci. Here, we report the identification of CYL5614, derived from strain Becker, with a mutation that affects the expression of type 8 capsular polysaccharide (CP8), nuclease, alpha-toxin, coagulase, protease, and protein A. This novel locus, named mgr, was linked by transposon Tn917 and mapped by three-factorial transduction crosses. The region containing the mgr locus was cloned and sequenced. Deletion mutagenesis and genetic complementation showed that the locus consisted of one gene, mgrA. Interestingly, mgrA-null mutants exhibited a phenotype opposite to that of CYL5614. This was due to a T-to-C mutation upstream of mgrA that resulted in a four- to eightfold increase in mgrA transcription in strain CYL5614. Thus, these results indicate that mgrA is an activator of CP8 and nuclease but a repressor of alpha-toxin, coagulase, protease, and protein A. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the mgr locus profoundly affected extracellular protein production, suggesting that the locus may regulate many other genes as well. The translated MgrA protein has a region of significant homology, which includes the helix-turn-helix DNA-binding motif, with the Escherichia coli MarR family of transcriptional regulators. Northern slot blot analyses suggested that mgr affected CP8, alpha-toxin, nuclease, and protein A at the transcriptional level.

Download Full-text

ClpC affects the intracellular survival capacity of Staphylococcus aureus in non-professional phagocytic cells

Scientific Reports ◽

10.1038/s41598-019-52731-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Gubesh Gunaratnam ◽

Lorena Tuchscherr ◽

Mohamed I. Elhawy ◽

Ralph Bertram ◽

Janina Eisenbeis ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Protein A ◽

Intracellular Survival ◽

Host Cells ◽

Virulence Determinants ◽

Phagocytic Cells ◽

Survival Capacity ◽

Shock Proteins ◽

Mutant Cells

Abstract Invasion and persistence of bacteria within host cells requires that they adapt to life in an intracellular environment. This adaptation induces bacterial stress through events such as phagocytosis and enhanced nutrient-restriction. During stress, bacteria synthesize a family of proteins known as heat shock proteins (HSPs) to facilitate adaptation and survival. Previously, we determined the Staphylococcus aureus HSP ClpC temporally alters bacterial metabolism and persistence. This led us to hypothesize that ClpC might alter intracellular survival. Inactivation of clpC in S. aureus strain DSM20231 significantly enhanced long-term intracellular survival in human epithelial (HaCaT) and endothelial (EA.hy926) cell lines, without markedly affecting adhesion or invasion. This phenotype was similar across a genetically diverse collection of S. aureus isolates, and was influenced by the toxin/antitoxin encoding locus mazEF. Importantly, MazEF alters mRNA synthesis and/or stability of S. aureus virulence determinants, indicating ClpC may act through the mRNA modulatory activity of MazEF. Transcriptional analyses of total RNAs isolated from intracellular DSM20231 and isogenic clpC mutant cells identified alterations in transcription of α-toxin (hla), protein A (spa), and RNAIII, consistent with the hypothesis that ClpC negatively affects the intracellular survival of S. aureus in non-professional phagocytic cells, via modulation of MazEF and Agr.

Download Full-text

Evaluation of IgY capture ELISA for sensitive detection of Alpha hemolysin of Staphylococcus aureus without staphylococcal protein A interference

Journal of Immunological Methods ◽

10.1016/j.jim.2013.02.004 ◽

2013 ◽

Vol 391 (1-2) ◽

pp. 31-38 ◽

Cited By ~ 34

Author(s):

Prakash Kudumala Reddy ◽

Aravind Shekar ◽

Joseph Jeyabalaji Kingston ◽

Murali Harishchandra Sripathy ◽

Harshvardhan Batra

Keyword(s):

Staphylococcus Aureus ◽

Protein A ◽

Sensitive Detection ◽

Staphylococcal Protein A ◽

Staphylococcal Protein ◽

Capture Elisa ◽

Alpha Hemolysin

Download Full-text

Alpha-hemolysin of Staphylococcus aureus impairs thrombus formation

10.1101/2021.11.11.468205 ◽

2021 ◽

Author(s):

Kristin Jahn ◽

Stefan Handtke ◽

Raghavendra Palankar ◽

Thomas P Kohler ◽

Jan Wesche ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Thrombus Formation ◽

Bloodstream Infections ◽

Human Platelets ◽

Virulence Determinants ◽

Aortic Valves ◽

Alpha Hemolysin ◽

Immune Globulins ◽

The Stability ◽

Systemic Infections

Toxins are key virulence determinants of pathogens and can impair the function of host immune cells including platelets. Insights into pathogen toxin interference with platelets will be pivotal to improve treatment of patients with bacterial bloodstream infections. In this study, we deciphered the effects of Staphylococcus aureus toxins alpha-hemolysin, LukAB, LukDE and LukSF on human platelets and compared the effects with the pore forming toxin pneumolysin of Streptococcus pneumoniae. In contrast to pneumolysin, alpha-hemolysin initially activates platelets as indicated by CD62P and alphaIIbeta3 integrin expression, but the resulting pores also induce alterations in the phenotype of platelets and induce apoptosis of platelets. The presence of small amounts of alpha-hemolysin (0.2 microgram/mL) in whole blood abrogates thrombus formation indicating that in systemic infections with S. aureus the stability of formed thrombi is impaired. This might be of high clinical relevance for S. aureus induced endocarditis of the aortic valves. Stabilizing the thrombi by inhibiting alpha-hemolysin induced impairment of platelets likely reduces the risk for septic (micro-)embolization. However, in contrast to pneumolysin, alpha-hemolysin induced platelets damage could not be neutralized by intravenous immune globulins. In contrast to alpha-hemolysin, S. aureus bi-component pore forming leukocidins LukAB, LukED and LukSF do not bind to platelets and had no significant effect on platelet activation and viability.

Download Full-text

SarZ Promotes the Expression of Virulence Factors and Represses Biofilm Formation by Modulating SarA and agr in Staphylococcus aureus

Infection and Immunity ◽

10.1128/iai.00859-08 ◽

2008 ◽

Vol 77 (1) ◽

pp. 419-428 ◽

Cited By ~ 35

Author(s):

Sandeep Tamber ◽

Ambrose L. Cheung

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Target Genes ◽

Protein A ◽

Dna Binding Protein ◽

Normal Flora ◽

Regulatory Pathways ◽

Downstream Target ◽

Alpha Hemolysin ◽

Important Regulator

ABSTRACT Staphylococcus aureus is a remarkably adaptable organism capable of multiple modes of growth in the human host, as a part of the normal flora, as a pathogen, or as a biofilm. Many of the regulatory pathways governing these modes of growth are centered on the activities of two regulatory molecules, the DNA binding protein SarA and the regulatory RNAIII effector molecule of the agr system. Here, we describe the modulation of these regulators and their downstream target genes by SarZ, a member of the SarA/MarR family of transcriptional regulators. Transcriptional and phenotypic analyses of a sarZ mutant demonstrated that the decreased transcription of mgrA and the agr RNAIII molecule was accompanied by increased transcription of spa (protein A) and downregulation of hla (alpha-hemolysin) and sspA (V8 protease) transcripts when compared to its isogenic parent. The decrease in protease activity was also associated with an increase in SarA expression. Consistent with an increase in SarA levels, the sarZ mutant displayed an enhanced ability to form biofilms. Together, our results indicate that SarZ may be an important regulator governing the dissemination phase of S. aureus infections, as it promotes toxin expression while repressing factors required for biofilm formation.

Download Full-text

Caenorhabditis elegans as a Model Host for Staphylococcus aureus Pathogenesis

Infection and Immunity ◽

10.1128/iai.71.4.2208-2217.2003 ◽

2003 ◽

Vol 71 (4) ◽

pp. 2208-2217 ◽

Cited By ~ 202

Author(s):

Costi D. Sifri ◽

Jakob Begun ◽

Frederick M. Ausubel ◽

Stephen B. Calderwood

Keyword(s):

Staphylococcus Aureus ◽

Caenorhabditis Elegans ◽

Regulatory System ◽

P38 Map Kinase ◽

Virulence Determinants ◽

Loss Of Function ◽

C Elegans ◽

Alpha Hemolysin ◽

Key Aspects ◽

Virulence Regulator

ABSTRACT Staphylococcus aureus, an important pathogen of humans and other warm-blooded animals, is also capable of killing the nematode Caenorhabditis elegans. Here, we show that C. elegans organisms that are fed S. aureus die over the course of several days in a process that is correlated with the accumulation of bacteria within the nematode digestive tract. Several S. aureus virulence determinants known or speculated to be important in mammalian pathogenesis, including the quorum-sensing global virulence regulatory system agr and the global virulence regulator sarA, the alternative sigma factor σB, alpha-hemolysin, and V8 serine protease, are required for full pathogenicity in nematodes. In addition, several defined C. elegans mutants were examined for susceptibility to S. aureus infection. Enhanced susceptibility to S. aureus killing was observed with loss-of-function mutations in the C. elegans genes esp-2/sek-1 and esp-8/nsy-1, which encode components of a conserved p38 MAP kinase signaling pathway involved in nematode defense against multiple pathogens. These results suggest that key aspects of S. aureus pathogenesis have been conserved, irrespective of the host, and that specific C. elegans host factors can alter susceptibility to this gram-positive human pathogen.

Download Full-text