Acinetobacter baumannii Regulates Its Stress Responses via the BfmRS Two-Component Regulatory System.

Acinetobacter baumannii is a common nosocomial pathogen that utilizes numerous mechanisms to aid its survival in both the environment and the host. Coordination of such mechanisms requires an intricate regulatory network. We report here that A. baumannii can directly regulate several stress-related pathways via the two-component regulatory system BfmRS. Similar to previous studies, results from transcriptomic analysis showed that mutation of the BfmR response regulator causes dysregulation of genes required for the oxidative stress response, the osmotic stress response, the misfolded protein/heat shock response, Csu pilus/fimbria production, and capsular polysaccharide biosynthesis. We also found that the BfmRS system is involved in controlling siderophore biosynthesis and transport, and type IV pili production. We provide evidence that BfmR binds to various stress-related promoter regions and show that BfmR alone can directly activate transcription of some stress-related genes. Additionally, we show that the BfmS sensor kinase acts as a BfmR phosphatase to negatively regulate BfmR activity. This work highlights the importance of the BfmRS system in promoting survival of A. baumannii. IMPORTANCE Acinetobacter baumannii is a nosocomial pathogen that has extremely high rates of multidrug resistance. This organism's ability to endure stressful conditions is a key part of its ability to spread in the hospital environment and cause infections. Unlike other members of the gammaproteobacteria, A. baumannii does not encode a homolog of the RpoS sigma factor to coordinate its stress response. Here, we demonstrate that the BfmRS two-component system directly controls the expression of multiple stress resistance genes. Our findings suggest that BfmRS is central to a unique scheme of general stress response regulation by A. baumannii.

2-Aminoimidazole Analogs Target PhoP Altering DNA Binding Activity and Affect Outer Membrane Stability in Gram-Negative Bacteria.

Multidrug-resistant bacteria cause immense public health concerns as once effective antibiotics no longer work against even common infections. Concomitantly, there has been a decline in the discovery of new antibiotics, and the current global clinical pipeline is woefully inadequate, especially against resistant Gram-negative bacteria. One major contribution to Gram-negative resistance is the presence of a protective outer membrane. Consequently, an appealing option for tackling resistance is to adversely affect that outer membrane. With that in mind, we define the response regulator PhoP as a target for new 2-aminoimidazole compounds and show that they affect the integrity of the outer membrane in resistant strains of Escherichia coli and Klebsiella pneumoniae. We also provide empirical evidence for the 2-aminoimidazole mechanism of action.

CsrA Supports both Environmental Persistence and Host-Associated Growth of Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic and frequently multidrug-resistant Gram-negative bacterial pathogen that primarily infects critically ill individuals. Indirect transmission from patient to patient in hospitals can drive infections, supported by this organism's abilities to persist on dry surfaces and rapidly colonize susceptible individuals. To investigate how A. baumannii survives on surfaces, we cultured A. baumannii in liquid media for several days and then analyzed isolates that lost the ability to survive drying. One of these isolates carried a mutation that affected the gene encoding the carbon storage regulator CsrA. As we began to examine the role of CsrA in A. baumannii, we observed that the growth of ΔcsrA mutant strains was inhibited in the presence of amino acids. The ΔcsrA mutant strains had a reduced ability to survive drying and to form biofilms but an improved ability to tolerate increased osmolarity compared with the wild type. We also examined the importance of CsrA for A. baumannii virulence. The ΔcsrA mutant strains had a greatly reduced ability to kill Galleria mellonella larvae, could not replicate in G. mellonella hemolymph, and also had a growth defect in human serum. Together, these results show that CsrA is essential for the growth of A. baumannii on host-derived substrates and is involved in desiccation tolerance, implying that CsrA controls key functions involved in the transmission of A. baumannii in hospitals.

Structure of the Acinetobacter baumannii PmrA receiver domain and insights into clinical mutants affecting DNA binding and promoting colistin resistance.

Acinetobacter baumannii is an insidious emerging nosocomial pathogen that has developed resistance to all available antimicrobials, including the last resort antibiotic, colistin. Colistin resistance often occurs due to mutations in the PmrAB two-component regulatory system. To better understand the regulatory mechanisms contributing to colistin resistance, we have biochemically characterized the A. baumannii PmrA response regulator. Initial DNA-binding analysis shows that A. baumannii PmrA bound to the Klebsiella pneumoniae PmrA box motif. This prompted analysis of the putative A. baumannii PmrAB regulon that indicated that the A. baumannii PmrA consensus box is 5'-HTTAAD N5 HTTAAD. Additionally, we provide the first structural information for the A. baumannii PmrA N-terminal domain through X-ray crystallography and we present a full-length model using molecular modelling. From these studies, we were able to infer the effects of two critical PmrA mutations, PmrA::I13M and PmrA::P102R, both of which confer increased colistin resistance. Based on these data, we suggest structural and dynamic reasons for how these mutations can affect PmrA function and hence encourage resistive traits. Understanding these mechanisms will aid in the development of new targeted antimicrobial therapies. Graphical Abstract.

PsrA controls the synthesis of the Pseudomonas aeruginosa quinolone signal via repression of the FadE homolog, PA0506.

Pseudomonas aeruginosa is a ubiquitous, Gram-negative opportunistic pathogen that can cause disease in various sites within the human body. This bacterium is a major source of nosocomial infections that are often difficult to treat due to high intrinsic antibiotic resistance and coordinated virulence factor production. P. aeruginosa utilizes three cell-to-cell signaling systems to regulate numerous genes in response to cell density. One of these systems utilizes the small molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal [PQS]) as a signal that acts as a co-inducer for the transcriptional regulator PqsR. Quinolone signaling is required for virulence in multiple infection models, and PQS is produced during human infections, making this system an attractive target for potential drug development. In this study we have examined the role of a TetR-type transcriptional regulator, PsrA, in the regulation of PQS production by P. aeruginosa. Previous studies showed that PsrA regulates genes of the fatty acid ß-oxidation pathway, including PA0506, which encodes a FadE homolog. In this report, we show that deletion of psrA resulted in a large decrease in PQS production and that co-deletion of PA0506 allowed PQS production to be restored to a wild type level. We also found that PQS production could be restored to the psrA mutant by the addition of oleic or octanoic acid. Taken together, our data suggest that psrA positively affects PQS production by repressing the transcription of PA0506, which leads to a decrease in the conversion of acyl-CoA compounds to enoyl-CoA compounds, thereby allowing some octanoyl-CoA to escape the ß-oxidation pathway and serve as a PQS precursor.