A Putative Cro-Like Repressor Contributes to Arylomycin Resistance in Staphylococcus aureus

ABSTRACTAntibiotic-resistant bacteria are a significant public health concern and motivate efforts to develop new classes of antibiotics. One such class of antibiotics is the arylomycins, which target type I signal peptidase (SPase), the enzyme responsible for the release of secreted proteins from their N-terminal leader sequences. Despite the essentiality, conservation, and relative accessibility of SPase, the activity of the arylomycins is limited against some bacteria, including the important human pathogenStaphylococcus aureus. To understand the origins of the limited activity againstS. aureus, we characterized the susceptibility of a panel of strains to two arylomycin derivatives, arylomycin A-C16and its more potent analog arylomycin M131. We observed a wide range of susceptibilities to the two arylomycins and found that resistant strains were sensitized by cotreatment with tunicamycin, which inhibits the first step of wall teichoic acid synthesis. To further understand howS. aureusresponds to the arylomycins, we profiled the transcriptional response ofS. aureusNCTC 8325 to growth-inhibitory concentrations of arylomycin M131 and found that it upregulates the cell wall stress stimulon (CWSS) and an operon consisting of a putative transcriptional regulator and three hypothetical proteins. Interestingly, we found that mutations in the putative transcriptional regulator are correlated with resistance, and selection for resistanceex vivodemonstrated that mutations in this gene are sufficient for resistance. The results begin to elucidate howS. aureuscopes with secretion stress and how it evolves resistance to the inhibition of SPase.

Download Full-text

An Alternative Terminal Step of the General Secretory Pathway in Staphylococcus aureus

mBio ◽

10.1128/mbio.01178-15 ◽

2015 ◽

Vol 6 (4) ◽

Cited By ~ 6

Author(s):

Arryn Craney ◽

Melissa M. Dix ◽

Ramkrishna Adhikary ◽

Benjamin F. Cravatt ◽

Floyd E. Romesberg

Keyword(s):

Staphylococcus Aureus ◽

Protein Trafficking ◽

Secretory Pathway ◽

Signal Peptidase ◽

Type I ◽

Wild Type ◽

Content Type ◽

Intrinsic Signals ◽

Translocated Proteins ◽

A Site

ABSTRACT Type I signal peptidase (SPase) is essential for viability in wild-type bacteria because the terminal step of the bacterial general secretory pathway requires its proteolytic activity to release proteins from their membrane-bound N-terminal leader sequences after translocation across the cytoplasmic membrane. Here, we identify the Staphylococcus aureus operon ayrRABC (SA0337 to SA0340) and show that once released from repression by AyrR, the protein products AyrABC together confer resistance to the SPase inhibitor arylomycin M131 by providing an alternate and novel method of releasing translocated proteins. Thus, the derepression of ayrRABC allows cells to bypass the essentiality of SPase. We demonstrate that AyrABC functionally complements SPase by mediating the processing of the normally secreted proteins, albeit in some cases with reduced efficiency and either without cleavage or via cleavage at a site N-terminal to the canonical SPase cleavage site. Thus, ayrRABC encodes a secretion stress-inducible alternate terminal step of the general secretory pathway. IMPORTANCE Addressing proteins for proper localization within or outside a cell in both eukaryotes and prokaryotes is often accomplished with intrinsic signals which mediate membrane translocation and which ultimately must be removed. The canonical enzyme responsible for the removal of translocation signals is bacterial type I signal peptidase (SPase), which functions at the terminal step of the general secretory pathway and is thus essential in wild-type bacteria. Here, we identify a four-gene operon in S. aureus that encodes an alternate terminal step of the general secretory pathway and thus makes SPase nonessential. The results have important implications for protein secretion in bacteria and potentially for protein trafficking in prokaryotes and eukaryotes in general.

Download Full-text

Transduction of Staphylococcal Cassette ChromosomemecElements between Strains of Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01058-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5233-5238 ◽

Cited By ~ 33

Author(s):

Caitlyn R. Scharn ◽

Fred C. Tenover ◽

Richard V. Goering

Keyword(s):

Staphylococcus Aureus ◽

Scientific Literature ◽

Methicillin Resistance ◽

Low Frequency ◽

Tetracycline Resistance ◽

Health Concern ◽

Type I ◽

Content Type ◽

Type Iv ◽

Lysogenic Phage

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) is a well-known public health concern. However, the means by which methicillin resistance genes are transferred among staphylococci in nature remains unknown. Older scientific literature suggests transduction as a means ofmecAtransfer, but the optimal conditions are reported to require plasmids and potentially a lysogenic phage. These reports preceded discovery of the staphylococcal cassette chromosomemec(SCCmec) elements. We undertook studies to confirm and clarify the conditions promoting transduction of SCCmecinS. aureuspopulations using well-characterized donor and recipient strains primarily of the USA300 lineage. Both bacteriophages 80α and 29 were capable of transducing SCCmectype IV and SCCmectype I to recipient strains ofS. aureus. Pulsed-field gel electrophoresis andmec-associateddrutyping were used to confirm the identity of the transductants. Transfer ofmecAvia transduction occurred at low frequency and required extended selection times formecAgene expression and the presence of a penicillinase plasmid in the recipient. However, interference with the process by clavulanic acid and the necessity of lysogeny with ϕ11 in the recipient or the presence of a small (4-kb) tetracycline resistance plasmid, as previously reported, were not confirmed. SCCmectransduction was occasionally associated with substantial deletions or truncation of SCCmecand the arginine catabolic metabolic element in USA300 recipients. Overall, these data clarify the conditions required for SCCmectransduction and document that rearrangements may occur during the process.

Download Full-text

Expression, Purification, and Biological Characterization of Babesia microti Apical Membrane Antigen 1

Infection and Immunity ◽

10.1128/iai.00168-15 ◽

2015 ◽

Vol 83 (10) ◽

pp. 3890-3901 ◽

Cited By ~ 16

Author(s):

Prasun Moitra ◽

Hong Zheng ◽

Vivek Anantharaman ◽

Rajdeep Banerjee ◽

Kazuyo Takeda ◽

...

Keyword(s):

Apical Membrane ◽

The United States ◽

Host Cells ◽

Health Concern ◽

Babesia Microti ◽

Type I ◽

Content Type ◽

Apical Membrane Antigen 1 ◽

Apical Membrane Antigen ◽

Extracellular Region

The intraerythrocytic apicomplexanBabesia microti, the primary causative agent of human babesiosis, is a major public health concern in the United States and elsewhere. Apicomplexans utilize a multiprotein complex that includes a type I membrane protein called apical membrane antigen 1 (AMA1) to invade host cells. We have isolated the full-lengthB. microtiAMA1 (BmAMA1) gene and determined its nucleotide sequence, as well as the amino acid sequence of the AMA1 protein. This protein contains an N-terminal signal sequence, an extracellular region, a transmembrane region, and a short conserved cytoplasmic tail. It shows the same domain organization as the AMA1 orthologs from piroplasm, coccidian, and haemosporidian apicomplexans but differs from all other currently known piroplasmida, including otherBabesiaandTheileriaspecies, in lacking two conserved cysteines in highly variable domain III of the extracellular region. Minimal polymorphism was detected in BmAMA1 gene sequences of parasite isolates from six babesiosis patients from Nantucket. Immunofluorescence microscopy studies showed that BmAMA1 is localized on the cell surface and cytoplasm near the apical end of the parasite. Native BmAMA1 from parasite lysate and refolded recombinant BmAMA1 (rBmAMA1) expressed inEscherichia colireacted with a mouse anti-BmAMA1 antibody using Western blotting.In vitrobinding studies showed that both native BmAMA1 and rBmAMA1 bind to human red blood cells (RBCs). This binding is trypsin and chymotrypsin treatment sensitive but neuraminidase independent. Incubation ofB. microtiparasites in human RBCs with a mouse anti-BmAMA1 antibody inhibited parasite growth by 80% in a 24-h assay. Based on its antigenically conserved nature and potential role in RBC invasion, BmAMA1 should be evaluated as a vaccine candidate.

Download Full-text

Antimicrobial Activity against Intraosteoblastic Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04359-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 2029-2036 ◽

Cited By ~ 36

Author(s):

Florent Valour ◽

Sophie Trouillet-Assant ◽

Natacha Riffard ◽

Jason Tasse ◽

Sacha Flammier ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Ex Vivo ◽

Joint Infection ◽

Treatment Strategies ◽

Bactericidal Effect ◽

Infection Model ◽

Content Type ◽

Intracellular Activity ◽

Mg63 Cells ◽

Choice Of Treatment

ABSTRACTAlthoughStaphylococcus aureuspersistence in osteoblasts, partly as small-colony variants (SCVs), can contribute to bone and joint infection (BJI) relapses, the intracellular activity of antimicrobials is not currently considered in the choice of treatment strategies for BJI. Here, antistaphylococcal antimicrobials were evaluated for their intraosteoblastic activity and their impact on the intracellular emergence of SCVs in anex vivoosteoblast infection model. Osteoblastic MG63 cells were infected for 2 h with HG001S. aureus. After killing the remaining extracellular bacteria with lysostaphin, infected cells were incubated for 24 h with antimicrobials at the intraosseous concentrations reached with standard therapeutic doses. Intracellular bacteria and SCVs were then quantified by plating cell lysates. A bactericidal effect was observed with fosfomycin, linezolid, tigecycline, oxacillin, rifampin, ofloxacin, and clindamycin, with reductions in the intracellular inocula of −2.5, −3.1, −3.9, −4.2, −4.9, −4.9, and −5.2 log10CFU/100,000 cells, respectively (P< 10−4). Conversely, a bacteriostatic effect was observed with ceftaroline and teicoplanin, whereas vancomycin and daptomycin had no significant impact on intracellular bacterial growth. Ofloxacin, daptomycin, and vancomycin significantly limited intracellular SCV emergence. Overall, ofloxacin was the only molecule to combine an excellent intracellular activity while limiting the emergence of SCVs. These data provide a basis for refining the choice of antibiotics to prioritise in the management of BJI, justifying the combination of a fluoroquinolone for its intracellular activity with an anti-biofilm molecule, such as rifampin.

Download Full-text

Differentiation of Community-Associated and Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolates and Identification of spa Types by Use of PCR and High-Resolution Melt Curve Analysis

Journal of Clinical Microbiology ◽

10.1128/jcm.02088-19 ◽

2020 ◽

Vol 58 (5) ◽

Cited By ~ 1

Author(s):

Seyed A. Ghorashi ◽

Jane Heller ◽

Quincy Zhang ◽

Shafi Sahibzada

Keyword(s):

Staphylococcus Aureus ◽

High Resolution ◽

Curve Analysis ◽

Health Concern ◽

Staphylococcal Protein A ◽

Visual Interpretation ◽

High Resolution Melt ◽

Methicillin Resistant ◽

Content Type ◽

Mrsa Strains

ABSTRACT Infections due to methicillin-resistant Staphylococcus aureus (MRSA) are present worldwide and represent a major public health concern. The capability of PCR followed by high-resolution melt (HRM) curve analysis for the detection of community-associated and livestock-associated MRSA strains and the identification of staphylococcal protein A (spa) locus was evaluated in 74 MRSA samples which were isolated from the environment, humans, and pigs on a single piggery. PCR-HRM curve analysis identified four spa types among MRSA samples and differentiated MRSA strains accordingly. A nonsubjective differentiation model was developed according to genetic confidence percentage values produced by tested samples, which did not require visual interpretation of HRM curve results. The test was carried out at different settings, and result data were reanalyzed and confirmed with DNA sequencing. PCR-HRM curve analysis proved to be a robust and reliable test for spa typing and can be used as a tool in epidemiological studies.

Download Full-text

Diversity and Genetic Basis for Carbapenem Resistance in a Coastal Marine Environment

Applied and Environmental Microbiology ◽

10.1128/aem.02939-19 ◽

2020 ◽

Vol 86 (10) ◽

Author(s):

Dewa A. P. Rasmika Dewi ◽

Barbara Götz ◽

Torsten Thomas

Keyword(s):

Aquatic Environment ◽

Carbapenem Resistance ◽

Clinical Samples ◽

Resistant Bacteria ◽

Clinical Environment ◽

Human Pathogens ◽

Environmental Distribution ◽

Content Type ◽

Pseudomonas Monteilii ◽

Wide Range

ABSTRACT Resistance to the “last-resort” antibiotics, such as carbapenems, has led to very few antibiotics being left to treat infections by multidrug-resistant bacteria. Spread of carbapenem resistance (CR) has been well characterized for the clinical environment. However, there is a lack of information about its environmental distribution. Our study reveals that CR is present in a wide range of Gram-negative bacteria in the coastal seawater environment, including four phyla, eight classes, and 30 genera. These bacteria were likely introduced into seawater via stormwater flows. Some CR isolates found here, such as Acinetobacter junii, Acinetobacter johnsonii, Brevundimonas vesicularis, Enterococcus durans, Pseudomonas monteilii, Pseudomonas fulva, and Stenotrophomonas maltophilia, are further relevant to human health. We also describe a novel metallo-β-lactamase (MBL) for marine Rheinheimera isolates with CR, which has likely been horizontally transferred to Citrobacter freundii or Enterobacter cloacae. In contrast, another MBL of the New Delhi type was likely acquired by environmental Variovorax isolates from Escherichia coli, Klebsiella pneumoniae, or Acinetobacter baumannii utilizing a plasmid. Our findings add to the growing body of evidence that the aquatic environment is both a reservoir and a vector for novel CR genes. IMPORTANCE Resistance against the “last-resort” antibiotics of the carbapenem family is often based on the production of carbapenemases, and this has been frequently observed in clinical samples. However, the dissemination of carbapenem resistance (CR) in the environment has been less well explored. Our study shows that CR is commonly found in a range of bacterial taxa in the coastal aquatic environment and can involve the exchange of novel metallo-β-lactamases from typical environmental bacteria to potential human pathogens or vice versa. The outcomes of this study contribute to a better understanding of how aquatic and marine bacteria can act as reservoirs and vectors for CR outside the clinical setting.

Download Full-text

In VitroActivities of Tedizolid and Linezolid against Gram-Positive Cocci Associated with Acute Bacterial Skin and Skin Structure Infections and Pneumonia

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00390-15 ◽

2015 ◽

Vol 59 (10) ◽

pp. 6262-6265 ◽

Cited By ~ 24

Author(s):

Ko-Hung Chen ◽

Yu-Tsung Huang ◽

Chun-Hsing Liao ◽

Wang-Hui Sheng ◽

Po-Ren Hsueh

Keyword(s):

Staphylococcus Aureus ◽

Dilution Method ◽

Agar Dilution Method ◽

Gram Positive ◽

Content Type ◽

Skin Structure ◽

Streptococcus Anginosus ◽

Wide Range ◽

Gram Positive Cocci

ABSTRACTTedizolid is a novel, expanded-spectrum oxazolidinone with potent activity against a wide range of Gram-positive pathogens. A total of 425 isolates of Gram-positive bacteria were obtained consecutively from patients with acute bacterial skin and skin structure infections (ABSSSIs) or pneumonia. These isolates included methicillin-susceptibleStaphylococcus aureus(MSSA) (n= 100), methicillin-resistantStaphylococcus aureus(MRSA) (n= 100),Streptococcus pyogenes(n= 50),Streptococcus agalactiae(n= 50),Streptococcus anginosusgroup (n= 75),Enterococcus faecalis(n= 50), and vancomycin-resistant enterococci (VRE) (Enterococcus faecium) (n= 50). The MICs of tedizolid and linezolid were determined by the agar dilution method. Tedizolid exhibited betterin vitroactivities than linezolid against MSSA (MIC90s, 0.5 versus 2 μg/ml), MRSA (MIC90s, 0.5 versus 2 μg/ml),S. pyogenes(MIC90s, 0.5 versus 2 μg/ml),S. agalactiae(MIC90s, 0.5 versus 2 μg/ml),Streptococcus anginosusgroup (MIC90s, 0.5 versus 2 μg/ml),E. faecalis(MIC90s, 0.5 versus 2 μg/ml), and VRE (MIC90s, 0.5 versus 2 μg/ml). The tedizolid MICs againstE. faecalis(n= 3) and VRE (n= 2) intermediate to linezolid (MICs, 4 μg/ml) were 1 μg/ml and 0.5 μg/ml, respectively. The tedizolid MIC90s against S. anginosus,S. constellatus, andS. intermediuswere 0.5, 1, and 0.5 μg/ml, respectively, and the rates of susceptibility based on the U.S. FDA MIC interpretive breakpoints to the isolates were 16%, 28%, and 72%, respectively. Tedizolid exhibited 2- to 4-fold betterin vitroactivities than linezolid against a variety of Gram-positive cocci associated with ABSSSIs and pneumonia. The lower susceptibilities of tedizolid against isolates ofS. anginosusandS. constellatusthan against those ofS. intermediusin Taiwan were noted.

Download Full-text

Enhanced Resistance to Bacterial Infection in Protegrin-1 Transgenic Mice

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01530-07 ◽

2008 ◽

Vol 52 (5) ◽

pp. 1812-1819 ◽

Cited By ~ 16

Author(s):

Queenie C. K. Cheung ◽

Patricia V. Turner ◽

Cheng Song ◽

De Wu ◽

Hugh Y. Cai ◽

...

Keyword(s):

Transgenic Mice ◽

Bacterial Infection ◽

Ectopic Expression ◽

Farm Animals ◽

Health Concern ◽

Resistant Bacteria ◽

Cell Counts ◽

Enhanced Resistance ◽

Wide Range

ABSTRACT Antibiotic-resistant bacteria have become a public health concern. It was suggested that one source of resistant pathogens may be food-producing animals. Alternative approaches are therefore needed to enhance the resistance of farm animals to bacterial infection. Protegrin-1 (PG-1) is a neutrophil-derived antimicrobial peptide that possesses activity against a wide range of bacteria and enveloped viruses. Here we report on the production of transgenic mice that ectopically expressed PG-1 and compare their susceptibilities to Actinobacillus suis infection with those of their wild-type (WT) littermates. Of the 126 mice that were challenged with A. suis, 87% of the transgenic mice survived, whereas 31% of their WT littermates survived. The PG-1 transgenic mice had significantly lower bacterial loads in their lungs and reduced numbers of pulmonary pathological lesions. The antimicrobial function of PG-1 was confirmed in vitro by using fibroblast cells isolated from the transgenic mice but not the WT mice. Moreover, differential blood cell counts in bronchoalveolar lavage fluid indicated greater number of neutrophils in PG-1 transgenic mice than in their WT littermates after bacterial challenge. Our data suggest that the ectopic expression of PG-1 in mice confers enhanced resistance to bacterial infection, laying the foundation for the development of livestock with improved resistance to infection.

Download Full-text

Surface Glycopolymers Are Crucial forIn VitroAnti-Wall Teichoic Acid IgG-Mediated Complement Activation and Opsonophagocytosis of Staphylococcus aureus

Infection and Immunity ◽

10.1128/iai.00767-15 ◽

2015 ◽

Vol 83 (11) ◽

pp. 4247-4255 ◽

Cited By ~ 20

Author(s):

Jong-Ho Lee ◽

Na-Hyang Kim ◽

Volker Winstel ◽

Kenji Kurokawa ◽

Jesper Larsen ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Teichoic Acid ◽

Complement C3 ◽

Glycerol Phosphate ◽

Glycosylation Pattern ◽

Wall Teichoic Acid ◽

Content Type ◽

Gram Positive Bacteria ◽

Capsule Production ◽

Cell Envelopes

ABSTRACTThe cell envelopes of many Gram-positive bacteria contain wall teichoic acids (WTAs).Staphylococcus aureusWTAs are composed of ribitol phosphate (RboP) or glycerol phosphate (GroP) backbones substituted withd-alanine andN-acetyl-d-glucosamine (GlcNAc) orN-acetyl-d-galactosamine (GalNAc). Two WTA glycosyltransferases, TarM and TarS, are responsible for modifying the RboP WTA with α-GlcNAc and β-GlcNAc, respectively. We recently reported that purified human serum anti-WTA IgG specifically recognizes β-GlcNAc of the staphylococcal RboP WTA and then facilitates complement C3 deposition and opsonophagocytosis ofS. aureuslaboratory strains. This prompted us to examine whether anti-WTA IgG can induce C3 deposition on a diverse set of clinicalS. aureusisolates. To this end, we compared anti-WTA IgG-mediated C3 deposition and opsonophagocytosis abilities using 13 different staphylococcal strains. Of note, the majority ofS. aureusstrains tested was recognized by anti-WTA IgG, resulting in C3 deposition and opsonophagocytosis. A minority of strains was not recognized by anti-WTA IgG, which correlated with either extensive capsule production or an alteration in the WTA glycosylation pattern. Our results demonstrate that the presence of WTAs with TarS-mediated glycosylation with β-GlcNAc in clinically isolatedS. aureusstrains is an important factor for induction of anti-WTA IgG-mediated C3 deposition and opsonophagocytosis.

Download Full-text