The opportunistic pathogen Pseudomonas aeruginosa often adapts to its host environment and causes recurrent nosocomial infections. The extracytoplasmic function (ECF) sigma factor enables bacteria to alter their gene expression in response to host environmental stimuli. Here, we report an ECF sigma factor, HxuI, which is rapidly induced once P. aeruginosa encounters the host. Host stresses such as iron limitation, oxidative stress, low oxygen, and nitric oxide induce the expression of hxuI. By combining RNA-seq and promoter-lacZ reporter fusion analysis, we reveal that HxuI can activate the expression of diverse metabolic and virulence pathways which are critical to P. aeruginosa infections, including iron acquisition, denitrification, pyocyanin synthesis, and bacteriocin production. Most importantly, overexpression of the hxuI in the laboratory strain PAO1 promotes its colonization in both murine lung and subcutaneous infections. Together, our findings show that HxuI, a key player in host stress-response, controls the in vivo adaptability and virulence of P. aeruginosa during infection. IMPORTANCE P. aeruginosa has a strong ability to adapt to diverse environments, making it capable of causing recurrent and multisite infections in clinics. Understanding host adaptive mechanisms plays an important guiding role in the development of new anti-infective agents. Here, we demonstrate that an ECFσ factor of P. aeruginosa response to the host-inflicted stresses, which promotes the bacterial in vivo fitness and pathogenicity. Furthermore, our findings may help explain the emergence of highly transmissible strains of P. aeruginosa and the acute exacerbations during chronic infections.

NrtR is a Nudix-related transcriptional regulator that is distributed among diverse bacteria and plays an important role in modulating bacterial intracellular NAD homeostasis. Previously, we showed that NrtR influences the T3SS expression and pathogenesis of Pseudomonas aeruginosa and demonstrated that NrtR mediates T3SS regulation through the cAMP/Vfr pathway. In the present study, we found that mutation of the nrtR gene leads to upregulation of the Hcp secretion island-I type VI secretion system (H1-T6SS). Further analysis revealed that mutation of the nrtR gene results in upregulation of regulatory RNAs (RsmY/RsmZ) that are known to control the H1-T6SS by sequestration of RsmA or RsmN. Simultaneous deletion of rsmY/rsmZ reduced the expression of H1-T6SS in the ΔnrtR mutant. In addition, overexpression of either rsmA or rsmN in ΔnrtR decreased H1-T6SS expression. Chromatin immunoprecipitation (ChIP)-Seq and electrophoretic mobility shift assay (EMSA) analyses revealed that NrtR directly binds to the promoters of rsmY, rsmZ and tssA1 (first gene of the H1-T6SS operon). Overall, the results from this study reveal the molecular details of NrtR-mediated regulation of H1-T6SS in P. aeruginosa. IMPORTANCE NrtR is a Nudix-related transcriptional regulator and controls the NAD cofactor biosynthesis in bacteria. P. aeruginosa NrtR binds to the intergenic region between nadD2 and pcnA to repress the expression of the two operons, therefore controlling the NAD biosynthesis. We have previously reported that NrtR controls T3SS expression via the cAMP/Vfr pathway in P. aeruginosa. However, the global regulatory function and direct binding targets of the NrtR remain elusive in P. aeruginosa. This study reveals novel direct regulatory targets of the NrtR in P. aeruginosa, elucidating the molecular mechanism of NrtR-mediated regulation of H1-T6SS.

Carbohydrate metabolism plays essential roles in energy generation and providing carbon skeletons for amino acid syntheses. In addition, carbohydrate metabolism has been shown to influence bacterial susceptibility to antibiotics and virulence. In this study, we demonstrate that citrate synthase gltA mutation can increase the expression of the type III secretion system (T3SS) genes and antibiotic tolerance in Pseudomonas aeruginosa. The stringent response is activated in the gltA mutant, and deletion of the (p)ppGpp synthetase gene relA restores the antibiotic tolerance and expression of the T3SS genes to wild-type level. We further demonstrate that the intracellular level of cAMP is increased by the stringent response in the gltA mutant, which increases the expression of the T3SS master regulator gene exsA. Overall, our results reveal an essential role of GltA in metabolism, antibiotic tolerance, and virulence, as well as a novel regulatory mechanism of the stringent response-mediated regulation of the T3SS in P. aeruginosa. IMPORTANCE Rising antimicrobial resistance imposes a severe threat to human health. It is urgent to develop novel antimicrobial strategies by understanding bacterial regulation of virulence and antimicrobial resistance determinants. The stringent response plays an essential role in virulence and antibiotic tolerance. Pseudomonas aeruginosa is an opportunistic pathogen that causes acute and chronic infections in humans. The bacterium produces an arsenal of virulence factors and is highly resistant to a variety of antibiotics. In this study, we provide evidence that citrate synthase GltA plays a critical role in P. aeruginosa metabolism and influences the antibiotic tolerance and virulence. We further reveal a role of the stringent response in the regulation of the antibiotic tolerance and virulence. The significance of this work is in elucidation of novel regulatory pathways that control both antibiotic tolerance and virulence in P. aeruginosa.

The evolution of bacterial antibiotic resistance is exhausting the list of currently used antibiotics and endangers those in the pipeline. The combination of antibiotics is a promising strategy that may suppress resistance development and/or achieve synergistic therapeutic effects. Eravacycline is a newly approved antibiotic that is effective against a variety of multidrug-resistant (MDR) pathogens. However, the evolution of resistance to eravacycline and strategies to suppress the evolution remain unexplored. Here, we demonstrated that a carbapenem-resistant Klebsiella pneumoniae clinical isolate quickly developed resistance to eravacycline, which is mainly caused by mutations in the gene encoding the Lon protease. The evolved resistant mutants display collateral sensitivities to β-lactam/β-lactamase inhibitor (BLBLI) combinations aztreonam/avibactam and ceftazidime-avibactam. Proteomic analysis revealed upregulation of the multidrug efflux system AcrA-AcrB-TolC and porin proteins OmpA and OmpU, which contributed to the increased resistance to eravacycline and susceptibility to BLBLIs, respectively. The combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam suppresses resistance development. We further demonstrated that eravacycline-resistant mutants evolved from an NDM-1-containing K. pneumoniae strain display collateral sensitivity to aztreonam/avibactam, and the combination of eravacycline with aztreonam/avibactam suppresses resistance development. In addition, the combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam displayed synergistic therapeutic effects in a murine cutaneous abscess model. Overall, our results revealed mechanisms of resistance to eravacycline and collateral sensitivities to BLBLIs and provided promising antibiotic combinations in the treatment of multidrug-resistant K. pneumoniae infections. IMPORTANCE The increasing bacterial antibiotic resistance is a serious threat to global public health, which demands novel antimicrobial medicines and treatment strategies. Eravacycline is a newly approved antibiotic that belongs to the tetracycline antibiotics. Here, we found that a multidrug-resistant Klebsiella pneumoniae clinical isolate rapidly developed resistance to eravacycline and the evolved resistant mutants displayed collateral sensitivity to antibiotics aztreonam/avibactam and ceftazidime-avibactam. We demonstrated that the combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam repressed resistance development and improved the treatment efficacies. We also elucidated the mechanisms that contribute to the increased resistance to eravacycline and susceptibility to aztreonam/avibactam and ceftazidime-avibactam. This work demonstrated the mechanisms of antibiotic resistance and collateral sensitivity and provided a new therapeutically option for effective antibiotic combinations.

Polymyxins are currently the last-resort antibiotics for the treatment of multidrug-resistant Gram-negative bacterial infections. To expand the understanding of the intrinsic resistance mechanism against polymyxins, a laboratory strain of Pseudomonas aeruginosa PAO1 was subjected to serial passage in the presence of sublethal doses of polymyxin B over a period of 30 days. By whole-genome sequencing of successively isolated polymyxin B-resistant isolates, we identified a frameshift mutation (L183fs) in the mvfR gene that further increased polymyxin resistance in the pmrB mutant background. A ΔmvfR mutation alone showed higher tolerance to polymyxin B due to altered lipopolysaccharide (LPS) on the surface of bacterial cells, which decreases its outer membrane permeability. In the ΔmvfR mutant, polymyxin B treatment caused the upregulation of rfaD, the gene involved in LPS core oligosaccharide synthesis, which is responsible for polymyxin tolerance. To the best of our knowledge, this is the first report of mvfR mutation conferring polymyxin resistance in P. aeruginosa via increased integrity of bacterial outer membrane. IMPORTANCE Antibiotic resistance imposes a considerable challenge for the treatment of P. aeruginosa infections. Polymyxins are the last-resort antibiotics for the treatment of multidrug-resistant P. aeruginosa infections. Understanding the development and mechanisms of bacterial resistance to polymyxins may provide clues for the development of new or improved therapeutic strategies effective against P. aeruginosa. In this study, using an in vitro evolution assay in combination with whole-genome sequencing, we demonstrated that MvfR controls tolerance to polymyxin B by regulating the rfaD gene in P. aeruginosa. Our results reveal a novel mechanism employed by P. aeruginosa in the defense against polymyxin antibiotics.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that can cause a variety of acute and chronic infections. The bacterium is highly resistant to numerous antibiotics. Murepavadin is a peptidomimetic antibiotic that blocks the function of P. aeruginosa lipopolysaccharide (LPS) transport protein D (LptD), thus inhibiting the insertion of LPS into the outer membrane. In this study, we demonstrated that sublethal concentrations of murepavadin enhance the bacterial outer membrane permeability. Proteomic analyses revealed the alteration of protein composition in bacterial inner and outer membranes following murepavadin treatment. The antisigma factor MucA was upregulated by murepavadin. In addition, the expression of the sigma E factor gene algU and the alginate synthesis gene algD was induced by murepavadin. Deletion of the algU gene reduces bacterial survival following murepavadin treatment, indicating a role of the envelope stress response in bacterial tolerance. We further demonstrated that murepavadin enhances the bactericidal activities of β-lactam antibiotics by promoting drug influx across the outer membrane. In a mouse model of acute pneumonia, the murepavadin-ceftazidime/avibactam combination showed synergistic therapeutic effect against P. aeruginosa infection. In addition, the combination of murepavadin with ceftazidime/avibactam slowed down the resistance development. In conclusion, our results reveal the response mechanism of P. aeruginosa to murepavadin and provide a promising antibiotic combination for the treatment of P. aeruginosa infections.IMPORTANCEThe ever increasing resistance of bacteria to antibiotics poses a serious threat to global public health. Novel antibiotics and treatment strategies are urgently needed. Murepavadin is a novel antibiotic that blocks the assembly of lipopolysaccharide (LPS) into the Pseudomonas aeruginosa outer membrane by inhibiting LPS transport protein D (LptD). Here, we demonstrated that murepavadin impairs bacterial outer membrane integrity, which induces the envelope stress response. We further found that the impaired outer membrane integrity increases the influx of β-lactam antibiotics, resulting in enhanced bactericidal effects. In addition, the combination of murepavadin and a β-lactam/β-lactamase inhibitor mixture (ceftazidime/avibactam) slowed down the resistance development of P. aeruginosa. Overall, this study demonstrates the bacterial response to murepavadin and provides a new combination strategy for effective treatment.

WCK 5222 (cefepime/zidebactam) is a β-lactam/β-lactamase inhibitor combination that is effective against a broad range of highly drug-resistant bacterial pathogens, including those producing metallo-β-lactamase. In this study, we isolated a multidrug-resistant Pseudomonas aeruginosa clinical strain that is resistant to a variety of β-lactam antibiotics and the ceftazidime-avibactam combination. A metallo-β-lactamase gene blaDIM-2 was identified on a self-transmissible megaplasmid in the strain, which confers the resistance to β-lactam antibiotics, leaving WCK 5222 potentially one of the last treatment resorts. In vitro passaging assay combined with whole-genome sequencing revealed mutations in the pbpA gene (encoding the zidebactam target protein PBP2) in the evolved resistant mutants. Among the mutations, a V516M mutation increased the bacterial virulence in a murine acute pneumonia model. Reconstitution of the mutations in the reference strain PAO1 verified their roles in the resistance to zidebactam and revealed their influences on cell morphology in the absence and presence of zidebactam. Microscale thermophoresis (MST) assays demonstrated that the mutations reduced the affinity between PBP2 and zidebactam to various extents. Overall, our results revealed that mutations in the pbpA gene might be a major cause of evolved resistance to WCK 5222 in clinical settings. IMPORTANCE Antibiotic resistance imposes a severe threat on human health. WCK 5222 is a β-lactam/β-lactamase inhibitor combination that is composed of cefepime and zidebactam. It is one of the few antibiotics in clinical trials that are effective against multidrug-resistant Pseudomonas aeruginosa, including those producing metallo-β-lactamases. Understanding the mechanisms and development of bacterial resistance to WCK 5222 may provide clues for the development of strategies to suppress resistant evolvement. In this study, we performed an in vitro passaging assay by using a multidrug-resistant P. aeruginosa clinical isolate. Our results revealed that mutations in the zidebactam target protein PBP2 play a major role in the bacterial resistance to WCK 5222. We further demonstrated that the mutations reduced the affinities between PBP2 and zidebactam and resulted in functional resistance of PBP2 to zidebactam.