scholarly journals Staphylococcus aureus Cell Wall Biosynthesis Modulates Bone Invasion and Osteomyelitis Pathogenesis

2021 ◽  
Vol 12 ◽  
Author(s):  
Elysia A. Masters ◽  
Gowrishankar Muthukrishnan ◽  
Lananh Ho ◽  
Ann Lindley Gill ◽  
Karen L. de Mesy Bentley ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Surface Protein ◽  
Implant Loosening ◽  
Penicillin Binding Protein ◽  
Cell Wall Synthesis ◽  
Transmission Electron ◽  
Surface Adhesins

Staphylococcus aureus invasion of the osteocyte lacuno-canalicular network (OLCN) is a novel mechanism of bacterial persistence and immune evasion in chronic osteomyelitis. Previous work highlighted S. aureus cell wall transpeptidase, penicillin binding protein 4 (PBP4), and surface adhesin, S. aureus surface protein C (SasC), as critical factors for bacterial deformation and propagation through nanopores in vitro, representative of the confined canaliculi in vivo. Given these findings, we hypothesized that cell wall synthesis machinery and surface adhesins enable durotaxis- and haptotaxis-guided invasion of the OLCN, respectively. Here, we investigated select S. aureus cell wall synthesis mutants (Δpbp3, Δatl, and ΔmreC) and surface adhesin mutants (ΔclfA and ΔsasC) for nanopore propagation in vitro and osteomyelitis pathogenesis in vivo. In vitro evaluation in the microfluidic silicon membrane-canalicular array (μSiM-CA) showed pbp3, atl, clfA, and sasC deletion reduced nanopore propagation. Using a murine model for implant-associated osteomyelitis, S. aureus cell wall synthesis proteins were found to be key modulators of S. aureus osteomyelitis pathogenesis, while surface adhesins had minimal effects. Specifically, deletion of pbp3 and atl decreased septic implant loosening and S. aureus abscess formation in the medullary cavity, while deletion of surface adhesins showed no significant differences. Further, peri-implant osteolysis, osteoclast activity, and receptor activator of nuclear factor kappa-B ligand (RANKL) production were decreased following pbp3 deletion. Most notably, transmission electron microscopy (TEM) imaging of infected bone showed that pbp3 was the only gene herein associated with decreased submicron invasion of canaliculi in vivo. Together, these results demonstrate that S. aureus cell wall synthesis enzymes are critical for OLCN invasion and osteomyelitis pathogenesis in vivo.

THE MODE OF ACTION OF THE ANTIBIOTIC PAROMOMYCIN ON STAPHYLOCOCCUS AUREUS

1966 ◽  
Vol 12 (6) ◽  
pp. 1157-1165 ◽  
Author(s):  
A. von Seefried ◽  
D. C. Jordan
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Binding Sites ◽  
Bactericidal Action ◽  
Plate Method ◽  
Vitro Binding ◽  
Resistant Mutants ◽  
Intracellular Binding

Paromomycin (Humatin, Parke Davis & Co.), a broad-spectrum aminoglycosidic antibiotic, inhibits the incorporation of amino acids into the trypsinsoluble protein fraction of Staphylococcus aureus 257. Protein synthesis is inhibited immediately, but the synthesis of cell-wall mucopeptide and alcohol-soluble proteins and lipids is not affected for approximately 35 min after antibiotic addition to actively growing cells. Paromomycin, at the ribosomal level, prevents the attachment of amino acyl-s-RNA and causes accumulation of m-RNA.Divalent cations (Ca++ and Mg++) antagonize the bactericidal action of paromomycin and interfere with the in vivo binding of the antibiotic on both the cell surface and the intracellular binding sites. In vitro binding to free ribosomes can be prevented and reversed by both monovalent and divalent cations.Using a "cylinder-plate" method, involving the displacement of antibiotic from cellular fractions by 0.2 M MgCl2, the antibiotic can be recovered from the ribosomes, cytoplasm, and the cell wall of paromomycin-sensitive S. aureus cells, but is not found in any of these fractions isolated from paromomycin-resistant cells developed from the sensitive parent strain. The resistant mutants apparently have lost the ability to adsorb and transport the antibiotic into the cell.

scholarly journals Impact of Bicarbonate-β-Lactam Exposures on Methicillin-Resistant Staphylococcus aureus (MRSA) Gene Expression in Bicarbonate-β-Lactam-Responsive vs. Non-Responsive Strains

Genes ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 1650
Author(s):  
Selvi C. Ersoy ◽  
Blake M. Hanson ◽  
Richard A. Proctor ◽  
Cesar A. Arias ◽  
Truc T. Tran ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Ex Vivo ◽  
In Vivo Models ◽  
Methicillin Resistant ◽  
Genetic Perturbations ◽  
Functional Relevance

Methicillin-resistant Staphylococcus aureus (MRSA) infections represent a difficult clinical treatment issue. Recently, a novel phenotype was discovered amongst selected MRSA which exhibited enhanced β-lactam susceptibility in vitro in the presence of NaHCO3 (termed ‘NaHCO3-responsiveness’). This increased β-lactam susceptibility phenotype has been verified in both ex vivo and in vivo models. Mechanistic studies to-date have implicated NaHCO3-mediated repression of genes involved in the production, as well as maturation, of the alternative penicillin-binding protein (PBP) 2a, a necessary component of MRSA β-lactam resistance. Herein, we utilized RNA-sequencing (RNA-seq) to identify genes that were differentially expressed in NaHCO3-responsive (MRSA 11/11) vs. non-responsive (COL) strains, in the presence vs. absence of NaHCO3-β-lactam co-exposures. These investigations revealed that NaHCO3 selectively repressed the expression of a cadre of genes in strain 11/11 known to be a part of the sigB-sarA-agr regulon, as well as a number of genes involved in the anchoring of cell wall proteins in MRSA. Moreover, several genes related to autolysis, cell division, and cell wall biosynthesis/remodeling, were also selectively impacted by NaHCO3-OXA exposure in the NaHCO3-responsive strain MRSA 11/11. These outcomes provide an important framework for further studies to mechanistically verify the functional relevance of these genetic perturbations to the NaHCO3-responsiveness phenotype in MRSA.

scholarly journals 2605. Mixed Subpopulation of Hemolytic and Non-Hemolytic Phenotype in Clinical Staphylococcus aureus Blood Isolates

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S905-S906
Author(s):  
Seongman Bae ◽  
Eunbeen Cho ◽  
Eunmi Yang ◽  
Hyeonji Seo ◽  
Eun Sil Kim ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Agar Plate ◽  
Surface Protein ◽  
Blood Agar Plate ◽  
Clinical Settings ◽  
Sheep Blood Agar Plate ◽  
Single Colony ◽  
Mixed Pattern

Abstract Background Agr is a key regulator that controls expression of secreted exoproteins and surface protein in Staphylococcus aureus. It has been reported that mixed status of two different phenotypes including agr functional and nonfunctional subpopulations can coexist in vitro and in vivo. However, data on the natural course and clinical implication of the mixed agr status is limited. We thus investigated the frequency and characteristics of the mixed agr in clinical settings. Methods We evaluated isogenic paired MRSA isolates collected from patients with persistent S. aureus bacteremia (SAB) between October 2010 and April 2016, and then prospectively performed surveillance for the presence of mixed agr function in MRSA isolates from patients with SAB between May 2016 and December 2017. The mixed agr status was evaluated by single colony evaluation on sheep blood agar plate containing RN4220 supernatant (β-hemolysin) (Figure 1). Cross-streaking with RN4220 and RNAIII measurement were performed to confirm the agr functionality of each of hemolytic and non-hemolytic colonies, separately. The expression levels of RNAIII, hla, and saeS/saeR were measured by real-time reverse transcription polymerase chain reaction. Results A total of 161 first blood isolates were collected during study period, and 6 isolates (4%) displayed mixed phenotype by single colony test. The mixed hemolytic pattern was observed in 5 out of 52 ST72 isolates (10%) and 1 out of 82 ST5 isolates (1%) (Figure 1). No difference was found in the genotypes between hemolytic and non-hemolytic colonies from each isolate. Of the 6 isolates, three lost mixed hemolytic features in the follow-up blood cultures (Table 1). One ST72 and one ST5 isolate showed agr mixed pattern determined by different RNAIII levels, but remaining four ST72 isolates had mixed hemolytic pattern due to different expression of hla correlated with saeS/saeR expression (Figure 2). Conclusion The mixture of agr function status among the clinical blood isolates of MRSA was rarely observed and isolates displaying heterogeneous hemolytic phenotype were largely due to differential expression of α-hemolysin. Further investigation is needed to unveil the clinical significance of mixture of different hemolytic phenotypes. Disclosures All authors: No reported disclosures.

scholarly journals YycH and YycI Regulate Expression of Staphylococcus aureus Autolysins by Activation of WalRK Phosphorylation

2020 ◽  
Vol 8 (6) ◽  
pp. 870
Author(s):  
Mike Gajdiss ◽  
Ian R. Monk ◽  
Ute Bertsche ◽  
Janina Kienemund ◽  
Tanja Funk ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Teichoic Acid ◽  
Regulatory System ◽  
Regulatory Function ◽  
Wall Teichoic Acid ◽  
Dependent Cell ◽  
Severe Infections

Staphylococcus aureus is a facultative pathogen that can encode numerous antibiotic resistance and immune evasion genes and can cause severe infections. Reduced susceptibility to last resort antibiotics such as vancomycin and daptomycin is often associated with mutations in walRK, an essential two-component regulatory system (TCS). This study focuses on the WalK accessory membrane proteins YycH and YycI and their influence on WalRK phosphorylation. Depletion of YycH and YycI by antisense RNA caused an impaired autolysis, indicating a positive regulatory function on WalK as has been previously described. Phosphorylation assays with full-length recombinant proteins in phospholipid liposomes showed that YycH and YycI stimulate WalK activity and that both regulatory proteins are needed for full activation of the WalK kinase. This was validated in vivo through examining the phosphorylation status of WalR using Phos-tag SDS-PAGE with a yycHI deletion mutant exhibiting reduced levels of phosphorylated WalR. In the yycHI knockdown strain, muropeptide composition of the cell wall was not affected, however, the wall teichoic acid content was increased. In conclusion, a direct modulation of WalRK phosphorylation activity by the accessory proteins YycH and YycI is reported both in vitro and in vivo. Taken together, our results show that YycH and YycI are important in the direct regulation of WalRK-dependent cell wall metabolism.

scholarly journals Immunoglobulins to Surface-Associated Biofilm Immunogens Provide a Novel Means of Visualization of Methicillin-Resistant Staphylococcus aureus Biofilms

2007 ◽  
Vol 73 (20) ◽  
pp. 6612-6619 ◽  
Author(s):  
Rebecca A. Brady ◽  
Jeff G. Leid ◽  
Jennifer Kofonow ◽  
J. William Costerton ◽  
Mark E. Shirtliff
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Protein Production ◽  
Specific Protein ◽  
Biofilm Growth ◽  
Methicillin Resistant ◽  
Associated Proteins ◽  
Space Function

ABSTRACT Antigens from the methicillin-resistant Staphylococcus aureus (MRSA) cell wall have been shown to be immunogenic in vivo and upregulated during biofilm growth. In this study, we created purified, recombinant forms of selected antigens and biofilm-upregulated, cell wall-associated proteins. These proteins were shown to cause a robust polyclonal immunoglobulin G (IgG) response when used to immunize rabbits. Antibodies against these recombinant proteins bound to the native forms of each protein as harvested from in vitro grown biofilms of MRSA, as determined both via Western blot analysis and immunofluorescence confocal microscopy. These IgGs could be utilized as imaging tools that localize to areas of specific protein production within a biofilm. This work illustrates that immunogenic, cell wall-associated, biofilm-upregulated proteins are promising for in vitro visualization of biofilm growth, architecture, and space-function relationships.

scholarly journals Fosfomycin plus β-Lactams as Synergistic Bactericidal Combinations for Experimental Endocarditis Due to Methicillin-Resistant and Glycopeptide-Intermediate Staphylococcus aureus

2015 ◽  
Vol 60 (1) ◽  
pp. 478-486 ◽  
Author(s):  
A. del Río ◽  
C. García-de-la-Mària ◽  
J. M. Entenza ◽  
O. Gasch ◽  
Y. Armero ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Standard Dose ◽  
High Dose ◽  
Penicillin Binding Protein ◽  
Beta Lactams ◽  
Methicillin Resistant ◽  
Content Type ◽  
Time Kill

ABSTRACTThe urgent need of effective therapies for methicillin-resistantStaphylococcus aureus(MRSA) infective endocarditis (IE) is a cause of concern. We aimed to ascertain thein vitroandin vivoactivity of the older antibiotic fosfomycin combined with different beta-lactams against MRSA and glycopeptide-intermediate-resistantS. aureus(GISA) strains. Time-kill tests with 10 isolates showed that fosfomycin plus imipenem (FOF+IPM) was the most active evaluated combination. In an aortic valve IE model with two strains (MRSA-277H and GISA-ATCC 700788), the following intravenous regimens were compared: fosfomycin (2 g every 8 h [q8h]) plus imipenem (1 g q6h) or ceftriaxone (2 g q12h) (FOF+CRO) and vancomycin at a standard dose (VAN-SD) (1 g q12h) and a high dose (VAN-HD) (1 g q6h). Whereas a significant reduction of MRSA-227H load in the vegetations (veg) was observed with FOF+IPM compared with VAN-SD (0 [interquartile range [IQR], 0 to 1] versus 2 [IQR, 0 to 5.1] log CFU/g veg;P= 0.01), no statistical differences were found with VAN-HD. In addition, FOF+IPM sterilized more vegetations than VAN-SD (11/15 [73%] versus 5/16 [31%];P= 0.02). The GISA-ATCC 700788 load in the vegetations was significantly lower after FOF+IPM or FOF+CRO treatment than with VAN-SD (2 [IQR, 0 to 2] and 0 [IQR, 0 to 2] versus 6.5 [IQR, 2 to 6.9] log CFU/g veg;P< 0.01). The number of sterilized vegetations after treatment with FOF+CRO was higher than after treatment with VAN-SD or VAN-HD (8/15 [53%] versus 4/20 [20%] or 4/20 [20%];P= 0.03). To assess the effect of FOF+IPM on penicillin binding protein (PBP) synthesis, molecular studies were performed, with results showing that FOF+IPM treatment significantly decreased PBP1, PBP2 (but not PBP2a), and PBP3 synthesis. These results allow clinicians to consider the use of FOF+IPM or FOF+CRO to treat MRSA or GISA IE.

scholarly journals Mutations inmmpLand in the Cell Wall Stress Stimulon Contribute to Resistance to Oxadiazole Antibiotics in Methicillin-Resistant Staphylococcus aureus

2014 ◽  
Vol 58 (10) ◽  
pp. 5841-5847 ◽  
Author(s):  
Qiaobin Xiao ◽  
Sergei Vakulenko ◽  
Mayland Chang ◽  
Shahriar Mobashery
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Wall Stress ◽  
Methicillin Resistant ◽  
Content Type ◽  
Cell Wall Stress ◽  
A Cell ◽  
Putative Member

ABSTRACTStaphylococcus aureusis a leading cause of hospital- and community-acquired infections, which exhibit broad resistance to various antibiotics. We recently disclosed the discovery of the oxadiazole class of antibiotics, which hasin vitroandin vivoactivities against methicillin-resistantS. aureus(MRSA). We report herein that MmpL, a putative member of the resistance, nodulation, and cell division (RND) family of proteins, contributes to oxadiazole resistance in theS. aureusstrain COL. Through serial passages, we generated twoS. aureusCOL variants that showed diminished susceptibilities to an oxadiazole antibiotic. The MICs for the oxadiazole against one strain (designatedS. aureusCOLI) increased reproducibly 2-fold (to 4 μg/ml), while against the other strain (S. aureusCOLR), they increased >4-fold (to >8 μg/ml, the limit of solubility). The COLRstrain was derived from the COLIstrain. Whole-genome sequencing revealed 31 mutations inS. aureusCOLR, of which 29 were shared with COLI. Consistent with our previous finding that oxadiazole antibiotics inhibit cell wall biosynthesis, we found 13 mutations that occurred either in structural genes or in promoters of the genes of the cell wall stress stimulon. Two unique mutations inS. aureusCOLRwere substitutions in two genes that encode the putative thioredoxin (SACOL1794) and MmpL (SACOL2566). A role formmpLin resistance to oxadiazoles was discerned from gene deletion and complementation experiments. To our knowledge, this is the first report that a cell wall-acting antibiotic selects for mutations in the cell wall stress stimulon and the first to implicate MmpL in resistance to antibiotics inS. aureus.

scholarly journals Successful Therapy of Experimental Endocarditis Caused by Vancomycin-Resistant Staphylococcus aureus with a Combination of Vancomycin and β-Lactam Antibiotics

2006 ◽  
Vol 50 (9) ◽  
pp. 2951-2956 ◽  
Author(s):  
Paige M. Fox ◽  
Russell J. Lampen ◽  
Katrina S. Stumpf ◽  
Gordon L. Archer ◽  
Michael W. Climo
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Rabbit Model ◽  
Bloodstream Infections ◽  
Aberrant Cell ◽  
Trial Treatment ◽  
Vancomycin Resistant ◽  
Vancomycin Treatment

ABSTRACT VRS1 is the first isolated strain of vancomycin-resistant Staphylococcus aureus (VRSA) found to carry the vanA gene complex previously described in Enterococcus. Under vancomycin pressure, VRS1 makes aberrant cell walls consisting of stem tetrapeptide and depsipeptide that lack the terminal d-Ala-d-Ala residues targeted by vancomycin. Previous data have suggested that this aberrant cell wall is not cross-linked by PBP2a, the enzyme responsible for cell wall transpeptidation in the presence of β-lactam antibiotics. We examined the efficacy of treating VRS1 with a combination of vancomycin and β-lactam antibiotics in vitro and in vivo. We found that the MIC of oxacillin for VRS1 decreased from >256 μg/ml to <1 μg/ml in the presence of vancomycin. Using the rabbit model of endocarditis, we treated VRS1-infected rabbits with nafcillin alone, vancomycin alone, or a combination of nafcillin and vancomycin. Treatment with nafcillin in combination with vancomycin cleared bloodstream infections within 24 h and sterilized 12/13 spleens (92%), as well as 8/13 kidneys (62%), following 3 days of treatment. Mean aortic valve vegetation counts were reduced 3.48 log10 CFU/g with the combination therapy (compared to untreated controls) and were significantly lower than with either vancomycin or nafcillin given alone. VRS1 was extremely virulent in this model, as no untreated rabbits survived the 3-day trial. Treatment of clinical infections due to VRSA with the combination of vancomycin and β-lactams may be an option, based on these results.

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