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Staphylococcus aureus possess three alternative σ factors, including a lone extracytoplasmic function σ factor, σS. Our group previously identified and characterized this element, mapping three sigS promoters, demonstrating its inducibility during stress and virulence inducing conditions and demonstrating a role for this factor in disease causation. In the present study, we identify a fourth promoter of the sigS operon, termed P4, located in a unique position internal to the sigS coding region. Transcriptional profiling revealed that expression from P4 is dominant to the three upstream promoters, particularly upon exposure to chemical stressors that elicit DNA damage and disrupt cell wall stability; each of which have previously been shown to stimulate sigS expression. Importantly, expression of this fourth promoter, followed by at least one or more of the upstream promoters, is induced during growth in serum and upon phagocytosis by RAW 264.7 murine macrophage-like cells. Finally, we demonstrate that a downstream gene, SACOL1829, bears a large 3΄ UTR that spans the sigS-SACOL1828 coding region, and may serve to compete with the P4 transcript to inhibit σS production. Collectively, these findings reveal a unique operon architecture for the sigS locus that indicates the potential for novel regulatory mechanisms governing its expression.

The vast majority of live attenuated typhoid vaccines are constructed from the Salmonella enterica serovar Typhi strain Ty2, which is devoid of a functioning alternative sigma factor, RpoS, due to the presence of a frameshift mutation. RpoS is a specialized sigma factor that plays an important role in the general stress response of a number of Gram-negative organisms, including Salmonella . Previous studies have demonstrated that this sigma factor is necessary for survival following exposure to acid, hydrogen peroxide, nutrient-limiting conditions, and starvation. In addition, studies with Salmonella enterica serovar Typhimurium and the mouse model of typhoid fever have shown that RpoS is important in colonization and survival within the infected murine host. We converted 4 clinically studied candidate typhoid vaccine strains derived from Ty2 [CVD908 -htrA , Ty800, and χ9639(pYA3493)] and the licensed live typhoid vaccine Ty21a (also derived from Ty2) to RpoS + and compared their abilities to withstand environmental stresses that may be encountered within the host to those of the RpoS - parent strains. The results of our study indicate that strains that contain a functional RpoS were better able to survive following stress and that they would be ideal for further development as safe, effective vaccines to prevent S . Typhi infections or as vectors in recombinant attenuated Salmonella vaccines (RASVs) designed to protect against other infectious disease agents in humans. The S . Typhi strains constructed and described here will be made freely available upon request, as will the suicide vector used to convert rpoS mutants to RpoS + . IMPORTANCE Recombinant attenuated Salmonella vaccines (RASVs) represent a unique prevention strategy to combating infectious disease because they utilize the ability of Salmonella to invade and colonize deep effector lymphoid tissues and deliver hetero- and homologous derived antigens at the lowest immunizing dose. Our recent clinical trial in human volunteers indicated that an RpoS + derivative of Ty2 was better at inducing immune responses than its RpoS - counterpart. In this study, we demonstrate that a functional RpoS allele is beneficial for developing effective live attenuated vaccines against S . Typhi or in using S . Typhi as a recombinant attenuated vaccine vector to deliver other protective antigens.

We previously reported a series of N 2 , N 4 -disubstituted quinazoline-2,4-diamines as dihydrofolate reductase inhibitors with potent in vitro and in vivo antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) strains. In this work, we extended our previous study to the Gram-negative pathogen Acinetobacter baumannii We determined that optimized N 2 , N 4 -disubstituted quinazoline-2,4-diamines are strongly antibacterial against multidrug-resistant A. baumannii strains when the 6-position is replaced with a halide or an alkyl substituent. Such agents display potent antibacterial activity, with MICs as low as 0.5 μM, while proving to be strongly bactericidal. Interestingly, these compounds also possess the potential for antibiofilm activity, eradicating 90% of cells within a biofilm at or near MICs. Using serial passage assays, we observed a limited capacity for the development of resistance toward these molecules (4-fold increase in MIC) compared to existing folic acid synthesis inhibitors, such as trimethoprim (64-fold increase) and sulfamethoxazole (128-fold increase). We also identified limited toxicity toward human cells, with 50% lethal doses (LD 50 s) of ≤23 μM for lead agents 4 and 5. Finally, we demonstrated that our lead agents have excellent in vivo efficacy, with lead agent 5 proving more efficacious than tigecycline in a murine model of A. baumannii infection (90% survival versus 66%), despite being used at a lower dose (2 versus 30 mg kg -1 ). Together, our results demonstrate that N 2 , N 4 -disubstituted quinazoline-2,4-diamines have strong antimicrobial and antibiofilm activities against both Gram-positive organisms and Gram-negative pathogens, suggesting strong potential for their development as antibacterial agents., (Copyright © 2017 American Society for Microbiology.)

Background: We previously identified an ECF sigma factor, σS, that is important in the stress and virulence response of Staphylococcus aureus. Transcriptional profiling of sigS revealed that it is differentially expressed in many laboratory and clinical isolates, suggesting the existence of regulatory networks that modulates its expression., Results: To identify regulators of sigS, we performed a pull down assay using S. aureus lysates and the sigS promoter. Through this we identified CymR as a negative effector of sigS expression. Electrophoretic mobility shift assays (EMSAs) revealed that CymR directly binds to the sigS promoter and negatively effects transcription. To more globally explore genetic regulation of sigS, a Tn551 transposon screen was performed, and identified insertions in genes that are involved in amino acid biosynthesis, DNA replication, recombination and repair pathways, and transcriptional regulators. In efforts to identify gain of function mutations, methyl nitro-nitrosoguanidine mutagenesis was performed on a sigS-lacZ reporter fusion strain. From this a number of clones displaying sigS upregulation were subject to whole genome sequencing, leading to the identification of the lactose phosphotransferase repressor, lacR, and the membrane histidine kinase, kdpD, as central regulators of sigS expression. Again using EMSAs we determined that LacR is an indirect regulator of sigS expression, while the response regulator, KdpE, directly binds to the promoter region of sigS., Conclusions: Collectively, our work suggests a complex regulatory network exists in S. aureus that modulates expression of the ECF sigma factor, σS.

A series of N(2),N(4)-disubstituted quinazoline-2,4-diamines has been synthesized and tested against multidrug resistant Staphylococcus aureus. A structure-activity and structure-property relationship study was conducted to identify new hit compounds. This study led to the identification of N(2),N(4)-disubstituted quinazoline-2,4-diamines with minimum inhibitory concentrations (MICs) in the low micromolar range in addition to favorable physicochemical properties. Testing of biological activity revealed limited potential for resistance to these agents, low toxicity, and highly effective in vivo activity, even with low dosing regimens. Collectively, these characteristics make this compound series a suitable platform for future development of antibacterial agents.

We present the draft genome sequence of methicillin-resistant Staphylococcus aureus strain CBD-635, from the USA100 lineage. This is a sepsis isolate obtained from Tampa General Hospital. This strain is spa type t003 and multilocus sequence typing (MLST) type ST5, and it has been used by our group in the study of novel antimicrobial chemotherapeutics.

Fluoroquinolone antibiotics have been a mainstay in the treatment of bacterial diseases. The most notable representative, ciprofloxacin, possesses potent antimicrobial activity; however, a rise in resistance to this agent necessitates development of novel derivatives to prolong the clinical lifespan of these antibiotics. Herein we have synthesized and analyzed the antimicrobial properties of a library of N-acylated ciprofloxacin analogues. We find that these compounds are broadly effective against Gram-positive and Gram-negative bacteria, with many proving more effective than the parental drug, and several possessing MICs ≤1.0 μg/ml against methicillin-resistant Staphylococcus aureus and Bartonella species. An analysis of spontaneous mutation frequencies reveals very low potential for resistance in MRSA compared to existing fluoroquinolones. Mode of action profiling reveals that modification of the piperazinyl nitrogen by acylation does not alter the effect of these molecules towards their bacterial target. We also present evidence that these N-acylated compounds are highly effective at killing intracellular bacteria, suggesting the suitability of these antibiotics for therapeutic treatment., (Copyright © 2012. Published by Elsevier Ltd.)

Previously we identified a novel component of the Staphylococcus aureus regulatory network, an extracytoplasmic function σ-factor, σ(S), involved in stress response and disease causation. Here we present additional characterization of σ(S), demonstrating a role for it in protection against DNA damage, cell wall disruption, and interaction with components of the innate immune system. Promoter mapping reveals the existence of three unique sigS start sites, one of which appears to be subject to autoregulation. Transcriptional profiling revealed that sigS expression remains low in a number of S. aureus wild types but is upregulated in the highly mutated strain RN4220. Further analysis demonstrates that sigS expression is inducible upon exposure to a variety of chemical stressors that elicit DNA damage, including methyl methanesulfonate and ciprofloxacin, as well as those that disrupt cell wall stability, such as ampicillin and oxacillin. Significantly, expression of sigS is highly induced during growth in serum and upon phagocytosis by RAW 264.7 murine macrophage-like cells. Phenotypically, σ(S) mutants display sensitivity to a broad range of DNA-damaging agents and cell wall-targeting antibiotics. Furthermore, the survivability of σ(S) mutants is strongly impacted during challenge by components of the innate immune system. Collectively, our data suggest that σ(S) likely serves dual functions within the S. aureus cell, protecting against both cytoplasmic and extracytoplasmic stresses. This further argues for its important, and perhaps novel, role in the S. aureus stress and virulence responses.

There is increasing demand to develop antimicrobial peptides (AMPs) as next generation antibiotic agents, as they have the potential to circumvent emerging drug resistance against conventional antibiotic treatments. Non-natural antimicrobial peptidomimetics are an ideal example of this, as they have significant potency and in vivo stability. Here we report for the first time the design of lipidated γ-AApeptides as antimicrobial agents. These lipo-γ-AApeptides show potent broad-spectrum activities against fungi and a series of Gram-positive and Gram-negative bacteria, including clinically relevant pathogens that are resistant to most antibiotics. We have analyzed their structure-function relationship and antimicrobial mechanisms using membrane depolarization and fluorescent microscopy assays. Introduction of unsaturated lipid chain significantly decreases hemolytic activity and thereby increases the selectivity. Furthermore, a representative lipo-γ-AApeptide did not induce drug resistance in S. aureus, even after 17 rounds of passaging. These results suggest that the lipo-γ-AApeptides have bactericidal mechanisms analogous to those of AMPs and have strong potential as a new class of novel antibiotic therapeutics.

Recent genomic studies have demonstrated that fungi can possess gene clusters encoding for the production of previously unobserved secondary metabolites. Activation of these attenuated or silenced genes to obtain either improved titers of known compounds or new ones altogether has been a subject of considerable interest. In our efforts to discover new chemotypes that are effective against infectious diseases, including malaria and methicillin-resistant Staphylococcus aureus (MRSA), we have isolated a strain of marine fungus, Leucostoma persoonii, that produces bioactive cytosporones. Epigenetic modifiers employed to activate secondary metabolite genes resulted in enhanced production of known cytosporones B (1, 360%), C (2, 580%) and E (3, 890%), as well as the production of the previously undescribed cytosporone R (4). Cytosporone E was the most bioactive, displaying an IC(90) of 13 µM toward Plasmodium falciparum, with A549 cytotoxicity IC(90) of 437 µM, representing a 90% inhibition therapeutic index (TI(90) = IC(90) A459/IC(90)P. falciparum) of 33. In addition, cytosporone E was active against MRSA with a minimal inhibitory concentration (MIC) of 72 µM and inhibition of MRSA biofilm at roughly half that value (minimum biofilm eradication counts, MBEC90, was found to be 39 µM).

Staphylococcus aureus is a bacterial pathogen that causes severe infections among humans. The increasing emergence of antibiotic resistance necessitates the development of new strategies to combat the spread of disease. One approach is photodynamic inactivation using porphyrin photosensitizers, which generate superoxide and other radicals in the presence of light, causing cell death via the oxidation of proteins and lipids. In this study, we analyzed a novel library of meso-substituted and metallated porphyrins for activity against multidrug-resistant S. aureus. From a library of 251 compounds, 51 showed antimicrobial activity, in three discrete classes of activity: those that functioned only in light, those that had toxicity only in darkness, and those that displayed activity regardless of illumination. We further demonstrated the broad-spectrum activity of these compounds against a variety of pathogens, including Bacillus anthracis, Enterococcus faecalis, and Escherichia coli. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) analyses of lead compounds (XPZ-263 and XPZ-271) revealed strong activity and killing towards methicillin-resistant S. aureus (MRSA) strains. An analysis of mutation frequencies revealed low incidences of resistance to lead compounds by E. coli and MRSA. Finally, an exploration of the underlying mechanism of action suggests that these compounds do not depend solely upon light-induced radical generation for toxicity, highlighting their potential for clinical applications.

We report the identification of a new class of antimicrobial peptidomimetics-γ-AApeptides with potent and broad-spectrum activity, including clinically-relevant strains that are unresponsive to most antibiotics. They are also not prone to select for drug-resistance.

Bacterial fatty acid synthesis (FAS) is a potentially important, albeit controversial, target for antimicrobial therapy. Recent studies have suggested that the addition of exogenous fatty acids (FAs) to growth media can circumvent the effects of FAS-targeting compounds on bacterial growth. Consequently, such agents may have limited in vivo applicability for the treatment of human disease, as free FAs are abundant within the body. Our group has previously developed N-thiolated β-lactams and found they function by interfering with FAS in select pathogenic bacteria, including MRSA. To determine if the FAS targeting activity of N-thiolated β-lactams can be abrogated by exogenous fatty acids, we performed MIC determinations for MRSA strains cultured with the fatty acids oleic acid and Tween 80. We find that, whilst the activity of the known FAS inhibitor triclosan is severely compromised by the addition of both oleic acid and Tween 80, exogenous FAs do not mitigate the antibacterial activity of N-thiolated β-lactams towards MRSA. Consequently, we propose that N-thiolated β-lactams are unique amongst FAS-inhibiting antimicrobials, as their effects are unimpeded by exogenous FAs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)