S. aureus is classified as a serious threat pathogen and is a priority that guides the discovery and development of new antibiotics. Despite growing knowledge of S. aureus metabolic capabilities, our understanding of its systems-level responses to different media types remains incomplete. Here, we develop a manually reconstructed genome-scale model (GEM-PRO) of metabolism with 3D protein structures for S. aureus USA300 str. JE2 containing 854 genes, 1,440 reactions, 1,327 metabolites and 673 3-dimensional protein structures. Computations were in 85% agreement with gene essentiality data from random barcode transposon site sequencing (RB-TnSeq) and 68% agreement with experimental physiological data. Comparisons of computational predictions with experimental observations highlight: 1) cases of non-essential biomass precursors; 2) metabolic genes subject to transcriptional regulation involved in Staphyloxanthin biosynthesis; 3) the essentiality of purine and amino acid biosynthesis in synthetic physiological media; and 4) a switch to aerobic fermentation upon exposure to extracellular glucose elucidated as a result of integrating time-course of quantitative exo-metabolomics data. An up-to-date GEM-PRO thus serves as a knowledge-based platform to elucidate S. aureus’ metabolic response to its environment.

The ability of Staphylococcus aureus to infect many different tissue sites is enabled, in part, by its transcriptional regulatory network (TRN) that coordinates its gene expression to respond to different environments. We elucidated the organization and activity of this TRN by applying independent component analysis to a compendium of 108 RNA-sequencing expression profiles from two S. aureus clinical strains (TCH1516 and LAC). ICA decomposed the S. aureus transcriptome into 29 independently modulated sets of genes (i-modulons) that revealed: 1) High confidence associations between 21 i-modulons and known regulators; 2) an association between an i-modulon and σS, whose regulatory role was previously undefined; 3) the regulatory organization of 65 virulence factors in the form of three i-modulons associated with AgrR, SaeR, and Vim-3; 4) the roles of three key transcription factors (CodY, Fur, and CcpA) in coordinating the metabolic and regulatory networks; and 5) a low-dimensional representation, involving the function of few transcription factors of changes in gene expression between two laboratory media (RPMI, cation adjust Mueller Hinton broth) and two physiological media (blood and serum). This representation of the TRN covers 842 genes representing 76% of the variance in gene expression that provides a quantitative reconstruction of transcriptional modules in S. aureus, and a platform enabling its full elucidation.

An increasing number of multidrug-resistant Acinetobacter baumannii (MDR-AB) infections have been reported worldwide, posing a threat to public health. The establishment of methods to elucidate the mechanism of action (MOA) of A. baumannii-specific antibiotics is needed to develop novel antimicrobial therapeutics with activity against MDR-AB. We previously developed bacterial cytological profiling (BCP) to understand the MOA of compounds in Escherichia coli and Bacillus subtilis. Given how distantly related A. baumannii is to these species, it was unclear to what extent it could be applied. Here, we implemented BCP as an antibiotic MOA discovery platform for A. baumannii. We found that the BCP platform can distinguish among six major antibiotic classes and can also subclassify antibiotics that inhibit the same cellular pathway but have different molecular targets. We used BCP to show that the compound NSC145612 inhibits the growth of A. baumannii via targeting RNA transcription. We confirmed this result by isolating and characterizing resistant mutants with mutations in the rpoB gene. Altogether, we conclude that BCP provides a useful tool for MOA studies of antibacterial compounds that are active against A. baumannii.

Background Multidrug-resistant (MDR) Acinetobacter baumannii infections have high mortality rates and few treatment options. Synergistic drug combinations may improve clinical outcome and reduce further emergence of resistance in MDR pathogens. Here we show an unexpected potent synergy of two translation inhibitors against the pathogen: commonly prescribed macrolide antibiotic azithromycin (AZM), widely ignored as a treatment alternative for invasive Gram-negative pathogens, and minocycline, among the current standard-of-care agents used for A. baumannii . Methods Media-dependent activities of AZM and MIN were evaluated in minimum inhibitory concentration assays and kinetic killing curves, alone or in combination, both in standard bacteriologic media (cation-adjusted Mueller-Hinton Broth) and more physiologic tissue culture media (RPMI), with variations of bicarbonate as a physiologic buffer. Synergy was calculated by fractional inhibitory concentration index (FICI). Therapeutic benefit of combining AZM and MIN was tested in a murine model of A. baumannii pneumonia. AZM + MIN synergism was probed mechanistically by bacterial cytological profiling (BCP), a quantitative fluorescence microscopy technique that identifies disrupted bacterial cellular pathways on a single cell level, and real-time kinetic measurement of translation inhibition via quantitative luminescence. AZM + MIN synergism was further evaluated vs. other contemporary high priority MDR bacterial pathogens. Findings Although two translation inhibitors are not expected to synergize, each drug complemented kinetic deficiencies of the other, speeding the initiation and extending the duration of translation inhibition as verified by FICI, BCP and kinetic luminescence markers. In an MDR A. baumannii pneumonia model, AZM + MIN combination therapy decreased lung bacterial burden and enhanced survival rates. Synergy between AZM and MIN was also detected vs. MDR strains of Gram-negative Klebsiella pneumoniae and Pseudomonas aeruginosa , and the leading Gram-positive pathogen methicillin-resistant Staphylococcus aureus . Interpretation As both agents are FDA approved with excellent safety profiles, clinical investigation of AZM and MIN combination regimens may immediately be contemplated for optimal treatment of A. baumannii and other MDR bacterial infections in humans. Fund National Institutes of Health U01 AI124326 (JP, GS, VN) and U54 HD090259 (GS, VN). IC was supported by the UCSD Research Training Program for Veterinarians T32 OD017863.

Pseudomonas aeruginosa is an important opportunistic pathogen that has become a serious problem due to increased rates of antibiotic resistance. Due to this along with a dearth in novel antibiotic development, especially against Gram-negative pathogens, new therapeutic strategies are needed to prevent a post-antibiotic era. Here we describe the importance of the vacJ/Mla pathway in resisting bactericidal actions of the host innate immune response. P. aeruginosa tn5 transposon mutants in genes from the VacJ/Mla pathway showed increased susceptibility to killing by the host cathelicidin antimicrobial peptide, LL-37 when compared to the wild-type parent strain. The P. aeruginosa vacJ− mutant demonstrated increased membrane permeability upon damage as well as sensitivity to killing in the presence of the detergent sodium dodecyl sulfate and the divalent cation chelator EDTA. When exposed to human whole blood and serum complement, the vacJ− mutant was killed more rapidly when compared to the wild-type parent strain and complemented mutant. Finally, in an in vivo mouse lung infection model, infection with the vacJ− mutant resulted in reduced mortality, lower bacterial burden, and reduced lung damage when compared to the wild-type strain. This study highlights the potential in therapeutically targeting the VacJ/Mla pathway in sensitizing P. aeruginosa to killing by the host innate immune response.