Our Science

For the last 25 years, my lab has been involved in the study of gene regulation and expression of virulence and resistance determinants in Staphylococcus aureus, a common cause of community-acquired and hospital-acquired infections. We have discovered the SarA locus in 1992 which was subsequently found to have agr-dependent and agr-independent regulation of virulence determinants in S. aureus.  Advances in bacterial genomics allow us to decipher that, besides protein-based regulation, sRNAs are also a critical resource in post-transcriptional control of virulence expression.  In contrast to protein-based control, sRNAs do not require new protein-based synthesis and hence enable rapid engagement in response to specific stimuli.

There are over 300 sRNAs in the S. aureus genome.  We recently discovered that SarA is a major repressor of sRNA. We recently examined the complete transcriptome of S. aureus lab strain HG003 and its isogenic sarA mutant with RNA-Seq. One of them, encoded within the sarA P3-P1 promoter, is a 196 nt sRNA called Teg49.  Teg49 can regulate the target gene via SarA-dependent and SarA-independent mechanisms.  RNA-Seq of Teg49 mutant vs. the sarA mutant and parent SH1000 showed that Teg49 regulates a small set of genes independent of SarA.  One of them is called SPIN secreted as a 102-residue preprotein which was cleaved to remove the signal peptide to yield the mature 73 residue protein.  SPIN protein was recently found to inhibit killing by azurophilic granules in human neutrophils. SPIN is thus an evasion factor that allows S. aureus to escape the powerful oxidative and lethal activity found in neutrophil.  Our goal is to characterize the Teg49-SPIN interaction as well as other sRNA involved in resistance of S. aureus to human neutrophil and other immune mechanisms.

Publication:

1. Samin Kim, Dindo Reyes, Marie Beaume, Patrice Francois and Ambrose Cheung. 2014. Contribution of teg49 sRNA in the 5’-upstream transcriptional region of sarA to virulence in Staphylococcus aureus. Infect. Immun. 2014. 82:4369-4379. PMID:25092913, PMCID:PMC4187880
2. AC Manna, S. Kim, L. Cengher, A. Corvaglia, S. Leo, P. Francois, AL Cheung. 2018. Small RNA Teg49 is derived from a sarA transcript and regulates virulence genes independent of SarA in Staphylococcus aureus. Infect Immun. Vol 86. e00635-17. doi:10.1128/IAI.00635-17. PMID:29133345. PMCID:PMC5778362

There are ~16 two component regulatory systems in S. aureus.  Among these, four of these systems lie adjacent to an ABC transporter that represents an efflux pump system consisting of a membrane permease and an ATPase.  We investigated one of these systems called GraRS-VraFG which is critical to resistance of host defense proteins (i.e. defensins). VraG is a conserved membrane permease with multiple transmembrane segments and a large extracellular loop (>200 residues). VraG, in conjunction with VraF which is an ATPase, comprise an efflux pump system which has not been well characterized with respect to bacterial defense against host innate immunity. Mutation of vraG and graS renders the resultant mutants sensitive to polymyxin B, a bacterial-derived cationic antimicrobial peptide standing as a proxy for human HDPs, a part of the host innate immunity. A vraG mutant also fails in their ability to up-regulate mprF and dltABCD, both of which contribute to an increase in surface positive charge to interfere with binding of positively charged HDPs.  Recent studies suggest that VraG complexes with GraS in a bacterial two-hybrid assay. The association is mostly based on charge-charge interaction.  The residue K380 is especially important for this interaction. Our goal is to understand the role of the VraG-GraS complex in resistance to HDPs both in vitro and in the context of human neutrophils and CF-derived alveolar macrophages.  we believe that a better understanding of the mechanistic aspect of this resistance will enable us to design novel strategies to block this interaction between HDPs and VraG-GraS to enable improved killing of bacteria by the native immune system.

Publication:

1. Soo-Jin Yang, Arnold S. Bayer, Nagendra N. Mishra, Michael Yeaman, Yan Q. Xiong and Ambrose L. Cheung. 2012. The role of the S. aureus GraRS two component regulatory system in sending of and resistance to cationic antimicrobial peptides.  Infect. Immun. 80:74-81. PMCID:PMC3255649
2. Junho Cho, Stephen K. Costa, Rachel M. Wierzbicki, William F.C. Rigby and Ambrose L. Cheung. The extracellular loop of the membrane permease VraG interacts with GraS to sense cationic antimicrobial peptides in Staphylococcus aureus. 2021.  PLoS Pathogens.

During the last few years, we have focused on the mechanism of antibiotic resistance, particularly in community-acquired and hospital-acquired methicillin resistant S. aureus (CA-and HA-MRSA).  More specifically, we have found that inactivation of pbp4 by cefoxitin in CA-MRSA strains has rendered them sensitive to oxacillin (16-fold decrease in MIC) even though the mecA gene is expressed normally.  Based on these data, we have devised a high-throughput combination screen of small compound libraries involving over 60,000 targeted compounds at the ICCB-Longwood Drug Screening Facility at Harvard Medical School where we searched for compounds that inhibit growth of S. aureus with ¼ MIC of oxacillin. We have characterized and published on two of these compounds, called DNAC-1 and DNAC-2, which have broad activities on their own against Gram+ priority pathogens (e.g. MRSA and Enterococcus).  Based on DNAC-2, we have synthesized several series of analogues, two of which, designated JRS3-56 (compound 1) and JRS4-32 (compound 2) in the 2^nd series, possess broad anti-Gram+ activity with little to no toxicity towards eukaryotic cells.  However, these compounds have poor predicted solubility with high cLogP, making oral formation difficult. We have since synthesized 3^rd generation analogues, focusing on compound 111 with low MIC and cLogP.  The SAR has also been tightened.  Pilot studies indicated that compound 111 likely targest the membrane, making them a new class of novel membrane-active agents against Gram+ priority agents beside daptomycin.  We did not find overt toxicity as defined by RBC lysis and LDH release from cultured human epithelial cells. The goal is to characterize these compounds through SAR to improve bactericidal activity in vitro and in vivo and drug-disposition properties including solubility, little or no toxicity and other desirable PK values.

Publication:

1. Dhanalakshmi R. Nair, João M. Monteiro, Guido Memmi, Jane Thanassi, Michael Pucci, Joseph Schwartzman, Mariana G. Pinho and Ambrose Cheung. 2015. Characterization of a novel small molecule that potentiates β-lactam activity against Gram positive and Gram negative pathogens.  AAC.  59:1876-1885. doi:10.1128/AAC.04164-14. PMCID: PMC4356822