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Murepavadin is a peptidomimetic that specifically targets the lipopolysaccharide transport protein LptD of Pseudomonas aeruginosa . Here, we found that murepavadin enhances the bactericidal efficacies of tobramycin and amikacin. We further demonstrated that murepavadin enhances bacterial respiration activity and subsequent membrane potential, which promotes intracellular uptake of aminoglycoside antibiotics. In addition, the murepavadin-amikacin combination displayed a synergistic bactericidal effect in a murine pneumonia model., Competing Interests: The authors declare no conflict of interest.

Carbon metabolism plays an essential role in bacterial pathogenesis and susceptibility to antibiotics. In Pseudomonas aeruginosa , Crc, Hfq, and a small RNA, CrcZ, are central regulators of carbon metabolism. By screening mutants of genes involved in carbon metabolism, we found that mutation of the tpiA gene reduces the expression of the type III secretion system (T3SS) and bacterial resistance to aminoglycoside antibiotics. TpiA is a triosephosphate isomerase that reversibly converts glyceraldehyde 3-phosphate to dihydroxyacetone phosphate, a key step connecting glucose metabolism with glycerol and phospholipid metabolisms. We found that mutation of the tpiA gene enhances the bacterial carbon metabolism, respiration, and oxidative phosphorylation, which increases the membrane potential and promotes the uptake of aminoglycoside antibiotics. Further studies revealed that the level of CrcZ is increased in the tpiA mutant due to enhanced stability. Mutation of the crcZ gene in the tpiA mutant background restored the expression of the T3SS genes and the bacterial resistance to aminoglycoside antibiotics. Overall, this study reveals an essential role of TpiA in the metabolism, virulence, and antibiotic resistance in P. aeruginosa IMPORTANCE The increase in bacterial resistance against antibiotics imposes a severe threat to public health. It is urgent to identify new drug targets and develop novel antimicrobials. Metabolic homeostasis of bacteria plays an essential role in their virulence and resistance to antibiotics. Recent studies demonstrated that antibiotic efficacies can be improved by modulating the bacterial metabolism. Pseudomonas aeruginosa is an important opportunistic human pathogen that causes various infections. The bacterium is intrinsically resistant to antibiotics. In this study, we provide clear evidence that TpiA (triosephosphate isomerase) plays an essential role in the metabolism of P. aeruginosa and influences bacterial virulence and antibiotic resistance. The significance of this work is in identifying a key enzyme in the metabolic network, which will provide clues as to the development of novel treatment strategies against infections caused by P. aeruginosa ., (Copyright © 2020 Xia et al.)

Background: Trans-translation is a ribosome rescue system that plays an important role in bacterial tolerance to environmental stresses. It is absent in animals, making it a potential treatment target. However, its role in antibiotic tolerance in Pseudomonas aeruginosa remains unknown., Methods: The role and activity of trans-translation during antibiotic treatment were examined with a trans-translation-deficient strain and a genetically modified trans-translation component gene, respectively. In vitro assays and murine infection models were used to examine the effects of suppression of trans-translation., Results: We found that the trans-translation system plays an essential role in P. aeruginosa tolerance to azithromycin and multiple aminoglycoside antibiotics. We further demonstrated that gentamicin could suppress the azithromycin-induced activation of trans-translation. Compared with each antibiotic individually, gentamicin and azithromycin combined increased the killing efficacy against planktonic and biofilm-associated P. aeruginosa cells, including a reference strain PA14 and its isogenic carbapenem-resistance oprD mutant, the mucoid strain FRD1, and multiple clinical isolates. Furthermore, the gentamicin-azithromycin resulted in improved bacterial clearance in murine acute pneumonia, biofilm implant, and cutaneous abscess infection models., Conclusions: Combination treatment with gentamicin and azithromycin is a promising strategy in combating P. aeruginosa infections., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Objectives: Bacterial persisters are a small subpopulation of cells that are highly tolerant of antibiotics and contribute to chronic and recalcitrant infections. Global gene expression in Pseudomonas aeruginosa persister cells and genes contributing to persister formation remain largely unknown. The objective of this study was to examine the gene expression profiles of the persister cells and those that regained growth in fresh medium, as well as to identify novel genes related to persister formation., Methods: P. aeruginosa persister cells and those that regrew in fresh medium were collected and subjected to RNA sequencing analysis. Genes up-regulated in the persister cells were overexpressed to evaluate their roles in persister formation. The functions of the persister-contributing genes were assessed with pulse-chase assay, affinity chromatography, fluorescence and electron microscopy, as well as a light-scattering assay., Results: An operon containing PA2282-PA2287 was up-regulated in the persister cells and down-regulated in the regrowing cells. PA2285 and PA2287 play key roles in persister formation. PA2285 and PA2287 were found to bind to RpoC and FtsZ, which are involved in transcription and cell division, respectively. Pulse-chase assays demonstrated inhibitory effects of PA2285 and PA2287 on the overall transcription. Meanwhile, light-scattering and microscopy assays demonstrated that PA2285 and PA2287 interfere with cell division by inhibiting FtsZ aggregation. PA2285 and PA2287 are conserved in pseudomonads and their homologous genes in Pseudomonas putida contribute to persister formation., Conclusions: PA2285 and PA2287 are novel bifunctional proteins that contribute to persister formation in P. aeruginosa., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Pseudomonas aeruginosa is an opportunistic bacterial pathogen and is intrinsically resistant to a variety of antibiotics. Oligoribonuclease (Orn) is a 3'-to-5' exonuclease that degrades nanoRNAs. The Orn controls biofilm formation by influencing the homeostasis of cyclic-di-GMP. Previously, we demonstrated that Orn contributes to the tolerance of P. aeruginosa to fluoroquinolone antibiotics by affecting the production of pyocins. In this study, we found that mutation in the orn gene reduces bacterial tolerance to aminoglycoside and β-lactam antibiotics, which is mainly due to a defective response to oxidative stresses. The major catalase KatA is downregulated in the orn mutant, and overexpression of the katA gene restores the bacterial tolerance to oxidative stresses and the antibiotics. We further demonstrated that Orn influenced the translation of the katA mRNA and narrowed down the region in the katA mRNA that is involved in the regulation of its translation. Therefore, our results revealed a novel role of the Orn in bacterial tolerance to oxidative stresses as well as aminoglycoside and β-lactam antibiotics., (Copyright © 2019 American Society for Microbiology.)

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen, which causes infectious disease in patients with cystic fibrosis and compromised immunity. P. aeruginosa is difficult to eradicate because of its intrinsic resistance to most traditional antibiotics as well as acquired resistance mechanisms after decades of antibiotic usage. A full understanding of the P. aeruginosa pathogenesis mechanisms is necessary for the development of novel prevention and treatment strategies. To identify novel vaccine candidates, here we comprehensively examined the expression levels of all the known outer membrane proteins in two P. aeruginosa strains in a murine acute pneumonia model. OprH was one of the most highly expressed proteins during infection. In addition, OprH is known to be highly immunogenic and accessible by host proteins. Thus, it was chosen as a vaccine candidate. To further identify vaccine candidates, 34 genes highly expressed during infection were evaluated for their contributions in virulence by testing individual transposon insertion mutants. Among them, fpvA, hasR , and foxA were found essential for bacterial virulence and therefore included in vaccine construction. Immunization with a mixture of FpvA, HasR, and FoxA rendered no protection, however, while immunization by OprH refolded in liposomes elicited specific opsonic antibodies and conferred protection against two lipopolysaccharide-heterologous P. aeruginosa strains (PA14 and PA103). Overall, by studying the expression profile of the P. aeruginosa outer membrane proteins during infection, we identified OprH as a potential vaccine candidate for the prevention of lung infection by P. aeruginosa .

Our understanding of the molecular mechanisms behind bacteria-phage interactions remains limited. Here we report that a small protein, SrpA, controls core cellular processes in response to phage infection and environmental signals in Pseudomonas aeruginosa. We show that SrpA is essential for efficient genome replication of phage K5, and controls transcription by binding to a palindromic sequence upstream of the phage RNA polymerase gene. We identify potential SrpA-binding sites in 66 promoter regions across the P. aeruginosa genome, and experimentally validate direct binding of SrpA to some of these sites. Using transcriptomics and further experiments, we show that SrpA, directly or indirectly, regulates many cellular processes including cell motility, chemotaxis, biofilm formation, pyocyanin synthesis and protein secretion, as well as virulence in a Caenorhabditis elegans model of infection. Further research on SrpA and similar proteins, which are widely present in many other bacteria, is warranted.

Pseudomonas aeruginosa causes various acute and chronic infections in humans. Treatment with azithromycin (AZM) has been shown to benefit patients with chronic P. aeruginosa infections. By binding to the exit tunnel of the 50S ribosome, AZM causes ribosome stalling and depletion of the intracellular tRNA pool. It has been shown that AZM is able to kill stationary-phase P. aeruginosa cells and repress quorum sensing-regulated virulence factors as well as swarming motility. In P. aeruginosa , the PA5470 gene encodes a putative peptide chain release factor whose expression is highly induced by macrolide antibiotics. However, its function remains unknown. Here, we found that overexpression of PA5470 increased bacterial tolerance against AZM and alleviated the repression of swarming motility. Ribosome pulldown assays revealed that PA5470 contributes to the release of ribosome stalled by AZM. We further demonstrate that overexpression of PA5470 counteracts AZM-mediated repression of the translation of the quorum sensing regulator RhlR. Overall, our results revealed a novel role of PA5470 in the bacterial response to AZM., (Copyright © 2018 American Society for Microbiology.)

As one of the first lines of host defense, monocytes play important roles in clearing infected microbes. The defensive response is triggered by recognition of diverse microbial moieties, including released factors, which modulate host immune responses to establish a harsh environment for clinically important bacterial pathogens. In this study, we found that the expression of PTX3, a soluble form of pattern recognition receptor, was induced by infection with live Pseudomonas aeruginosa or treatment of cells with its supernatant. P. aeruginosa GroEL, a homolog of heat shock protein 60, was identified as one of the factors responsible for inducing the expression of PTX3 in host cells. GroEL induced PTX3 expression by activating the Toll-like receptor 4 (TLR4)-dependent pathway via nuclear factor-kappa B (NF-κB), while simultaneously inhibiting expression of microRNA-9, which targets the PTX3 transcript. Finally, by acting as an opsonin, GroEL-induced PTX3 promoted the association and phagocytosis of Staphylococcus aureus into macrophages. These data suggest that the host defensive environment is supported by the production of PTX3 in response to GroEL, which thus has therapeutic potential for clearance of bacterial infections., (Copyright © 2017 American Society for Microbiology.)

DExD/H box RNA helicases play essential roles in various biological processes in prokaryotes and eukaryotes. By screening Pseudomonas aeruginosa strains with mutations in various DExD/H box helicase genes, we identified that deaD was required for bacterial cytotoxicity and virulence in a mouse acute pneumonia model. Compared to a wild-type strain and its complementation strain, the deaD mutant induced less production of proinflammatory cytokines, neutrophil infiltration and lung damage during infection. We further found that the RNA helicase activity of DeaD was required for the expression of type III secretion system (T3SS) genes. Overexpression of ExsA, a master activator of the T3SS, restored the expression of T3SS genes as well as the virulence of the deaD mutant, suggesting that the attenuated virulence of the deaD mutant was mainly due to the defective T3SS. Overall, our results reveal a role of DeaD in the virulence of P. aeruginosa., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pseudomonas aeruginosa is an opportunistic pathogen that causes acute and chronic infections in humans. Pyocins are bacteriocins produced by P. aeruginosa that are usually released through lysis of the producer strains. Expression of pyocin genes is negatively regulated by PrtR, which gets cleaved under SOS response, leading to upregulation of pyocin synthetic genes. Previously, we demonstrated that PrtR is required for the expression of type III secretion system (T3SS), which is an important virulence component of P. aeruginosa. In this study, we demonstrate that mutation in prtR results in reduced bacterial colonization in a mouse acute pneumonia model. Examination of bacterial and host cells in the bronchoalveolar lavage fluids from infected mice revealed that expression of PrtR is induced by reactive oxygen species (ROS) released by neutrophils. We further demonstrate that treatment with hydrogen peroxide or ciprofloxacin, known to induce the SOS response and pyocin production, resulted in an elevated PrtR mRNA level. Overexpression of PrtR by a tac promoter repressed the endogenous prtR promoter activity, and electrophoretic mobility shift assay revealed that PrtR binds to its own promoter, suggesting an autorepressive mechanism of regulation. A high level of PrtR expressed from a plasmid resulted in increased T3SS gene expression during infection and higher resistance against ciprofloxacin. Overall, our results suggest that the autorepression of PrtR contributes to the maintenance of a relatively stable level of PrtR, which is permissive to T3SS gene expression in the presence of ROS while increasing bacterial tolerance to stresses, such as ciprofloxacin, by limiting pyocin production.

Bacterial biofilms have been proposed to be a major factor contributing to the failure of chronic wounds to heal because of their increased tolerance to antimicrobial agents and the prolonged inflammation they cause. Phenotypic characteristics of bacterial biofilms vary depending on the substratum to which they attach, the nutritional environment, and the microorganisms within the biofilm community. To develop an ex vivo biofilm model that more closely mimics biofilms in chronic skin wounds, we developed an optimal procedure to grow mature biofilms on a central partial-thickness wound in 12-mm porcine skin explants. Chlorine gas produced optimal sterilization of explants while preserving histological properties of the epidermis and dermis. Pseudomonas aeruginosa and Staphylococcus aureus developed mature biofilms after 3 days that had dramatically increased tolerance to gentamicin and oxacillin (∼100× and 8,000× minimal inhibitory concentration, respectively) and to sodium hypochlorite (0.6% active chlorine). Scanning electron microscopy and confocal microscopy verified extensive exopolymeric biofilm structures on the explants. Despite a significant delay, a ΔlasI quorum-sensing mutant of P. aeruginosa developed biofilm as antibiotic-tolerant as wild-type after 3 days. This ex vivo model simulates growth of biofilms on skin wounds and provides an accurate model to assess effects of antimicrobial agents on mature biofilms., (© 2013 by the Wound Healing Society.)

Forced exogenous gene expression has been well characterized as an effective method for directing both cellular differentiation and dedifferentiation. However, transgene expression is not amenable for therapeutic application due to potential insertional mutagenesis. Protein-based techniques provide a safe alternative, but current protein delivery methods are quite limited by labor-intensive purification processes, low protein yield, and inefficient intracellular targeting. Such limitations may be overcome by using a naturally occurring bacterial protein injection system, called the type III secretion system (T3SS), which injects bacterial proteins directly into the eukaryotic cell cytoplasm. Using a genetically attenuated strain of Pseudomonas aeruginosa, we have previously described the ability of this system to easily deliver a high quantity of protein to both differentiated and pluripotent cells. MyoD is a key muscle regulatory factor, the overexpression of which is able to induce transdifferentiation of numerous cell types into functional myocytes. Here we demonstrate transient injection of MyoD protein by P. aeruginosa to be sufficient to induce myogenic conversion of mouse embryonic fibroblasts. In addition to clear morphological changes, muscle-specific gene expression has been observed both at mRNA and protein levels. These studies serve as a foundation for the bacterial delivery of transcription factors to efficiently modulate concentration-dependent and temporal activation of gene expression that directs cell fate without jeopardizing genomic integrity.

The type III secretion system (T3SS) is a complex nanomachine of many pathogenic gram-negative bacteria. It forms a proteinaceous channel that is inserted into the host eukaryotic cell membrane for injection of bacterial proteins that manipulate host cell signaling. However, few studies have focused on the effector-independent functions of the T3SS. Using a murine model of acute lung infection with Pseudomonas aeruginosa, an important human opportunistic pathogen, we compared the pathogenicity of mutant bacteria that lack all of the known effector toxins ( ΔSTY), with mutant bacteria that also lack the major translocator protein PopB (ΔSTY/ΔPopB) and so cannot form a functional T3SS channel in the host cell membrane. Mortality was higher among mice challenged with ΔSTY compared to mice challenged with ΔSTY/ΔPopB mutant bacteria. In addition, mice infected with ΔSTY showed decreased bacterial clearance from the lungs compared to those infected with ΔSTY/ΔPopB. Infection was in both cases associated with substantial killing of lung infiltrating macrophages. However, macrophages from ΔSTY-infected mice died by pro-inflammatory necrosis characterized by membrane permeabilization and caspase-1 mediated IL-1β production, whereas macrophages from ΔSTY/ΔPopB infected mice died by apoptosis, which is characterized by annexin V positive staining of the cell membrane and caspase-3 activation. This was confirmed in macrophages infected in vitro. These results demonstrate a T3SS effector toxin independent role for the T3SS, in particular the T3SS translocator protein PopB, in the pathogenicity of P. aeruginosa during acute lung infection.

The Pseudomonas aeruginosa quorum sensing (QS) system is controlled by the signal molecules acyl homoserine lactones (AHLs) that are synthesized from acyl enoyl-acyl carrier proteins (acyl-ACPs) provided by the fatty acid biosynthesis cycle. Pfm (PA2950), an enoyl-CoA reductase, has previously been shown to affect swimming mobility and fatty acid biosynthesis. In this report, we further show that pfm influences bacterial adherence to human cells. Microarray assay results suggest that pfm affects bacterial adherence through its influence on the QS system. Further experiments confirmed that the pfm mutant strain produces significantly less QS signal molecules than the corresponding wild-type strain. Using strains Escherichia coli DH5α(pECP64, lasB'-lacZ) and E. coli DH5α(pECP61.5, rhlA'-lacZ), biosensors for N-(3-oxododecanoyl) homoserine lactone (3O-C(12) -HSL) and N-butyryl homoserine lactone (C(4) -HSL), respectively, we found that pfm mutant strain produces decreased amounts of both signal molecules. Elastase activity and pyocyanin measurements further confirmed the reduced levels of 3O-C(12) -HSL and C(4) -HSL in the pfm mutant. Finally, bacterial virulence, as assessed by the Caenorhabditis elegans worm killing assay, is decreased in the pfm mutant. Taken together, these data indicate that pfm can be an important target for the control of P. aeruginosa infectivity., (2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen. Successful injection of virulence factors into host cells, evasion of phagocytosis and promotion of pathogenesis depend primarily on the function of type III secretion system (T3SS). A complex set of signaling pathways have been shown to modulate T3SS expression. In this review, a brief introduction is given on the composition, function, and molecular determinants that regulate P. aeruginosa T3SS gene expression.

Background: Bloodstream infections with Pseudomonas aeruginosa have been well-described in neonatal intensive care units (NICU) and have resulted in the temporary closure of some nurseries to new admissions. Nosocomial transmission of these infections has been verified by fingerprint analysis of the isolates. We utilized molecular fingerprinting to identify the source of bloodstream infections in an NICU and used this information to apply infection control measures that allowed the nursery to stay open and continue to accept referrals., Methods: In June 1998 three premature infants transferred to our hospital (Hospital A) from Hospitals B and C had bloodstream infections with P. aeruginosa. Subsequently one additional neonate transferred from Hospital B was colonized with P. aeruginosa. Random amplification of polymorphic deoxyribonucleic acid (RAPD) was performed on the four isolates. All transfers from Hospital B were cultured, and surveillance programs were instituted in Hospitals A and B. Targeted infection control measures for all transfers were implemented., Results: The four isolates were the same clone by RAPD. Investigation of the environment in Hospital A did not identify any source of the organism. Surveillance cultures on 49 neonates at Hospital A revealed only one patient colonized at an endotracheal tube. This patient was also a transfer from Hospital B. Results from Hospital B identified 4 of 40 (10%) neonates colonized. All isolates were clones identical with the bloodstream isolates from the neonates with bloodstream infections. Infection control measures for all babies transferred from Hospital B resulted in no new cases of P. aeruginosa bacteremia during the next 5 years., Conclusions: The use of molecular fingerprinting of isolates of P. aeruginosa allowed for a prompt and directed infection control plan to be implemented in Hospitals A and B. It also allowed the NICU in Hospital A to continue to accept referrals from other hospitals and to implement a targeted infection control plan for patients transferred from Hospital B.