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Ehrlichia chaffeensis infects and proliferates inside monocytes or macrophages and causes human monocytic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. After internalization, E. chaffeensis resides in specialized membrane-bound inclusions, E. chaffeensis -containing vesicles (ECVs), to evade from host cell innate immune responses and obtain nutrients. However, mechanisms exploited by host cells to inhibit E. chaffeensis growth in ECVs are still largely unknown. Here we demonstrate that host cells recognize E. chaffeensis Ech_1067, a penicillin-binding protein, and then upregulate the expression of PIT1, which is a phosphate transporter and transports phosphate from ECVs to the cytosol to inhibit bacterial growth. We found that host cells upregulate the PIT1 expression upon E. chaffeensis infection using transcriptome sequencing, qRT-PCR and Western blotting, and PIT1 is localized on the ECV membrane in infected THP-1 cells using confocal microscopy. Silence of PIT1 using shRNA enhances E. chaffeensis intracellular growth. Finally, we found that E. chaffeensis Ech_1067 induces the upregulation of PIT1 expression through the MyD88-NF-κB pathway using recombinant protein for stimulation and siRNA for silence. Our findings deepen the understanding of the innate immune responses of host cells to inhibit bacterial intracellular growth and facilitate the development of new therapeutics for HME.

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.

Pathogenic bacteria have devastating impacts on human health as a result of acquired antibiotic resistance and innate tolerance. Every class of our current antibiotic arsenal was initially discovered as growth-inhibiting agents that target actively replicating (individual, free-floating) planktonic bacteria. Bacteria are notorious for utilizing a diversity of resistance mechanisms to overcome the action of conventional antibiotic therapies and forming surface-attached biofilm communities enriched in (non-replicating) persister cells. To address problems associated with pathogenic bacteria, our group is developing halogenated phenazine (HP) molecules that demonstrate potent antibacterial and biofilm-eradicating activities through a unique iron starvation mode of action. In this study, we designed, synthesized, and investigated a focused collection of carbonate-linked HP prodrugs bearing a quinone trigger to target the reductive cytoplasm of bacteria for bioactivation and subsequent HP release. The quinone moiety also contains a polyethylene glycol group, which dramatically enhances the water-solubility properties of the HP-quinone prodrugs reported herein. We found carbonate-linked HP-quinone prodrugs 11 , 21-23 to demonstrate good linker stability, rapid release of the active HP warhead following dithiothreitol (reductive) treatment, and potent antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis , and Enterococcus faecalis . In addition, HP-quinone prodrug 21 induced rapid iron starvation in MRSA and S. epidermidis biofilms, illustrating prodrug action within these surface-attached communities. Overall, we are highly encouraged by these findings and believe that HP prodrugs have the potential to address antibiotic resistant and tolerant bacterial infections.

Escherichia coli (E. coli) cells are present in fecal materials that can be the main source for disease-causing agents in water. As a result, E. coli is recommended as a water quality indicator. We have developed an innovative platform to detect E. coli for monitoring water quality on-site by integrating paper-based sample preparation with nucleic acid isothermal amplification. The platform carries out bacterial lysis and DNA enrichment onto a paper pad through ball-based valves for fluid control, with no need of laboratory equipment, followed by loop-mediated isothermal amplification (LAMP) in a battery-operated coffee mug, and colorimetric detection. We have used the platform to detect E. coli in environmental water samples in about 1 h, with a limit of quantitation of 0.2 CFU/mL, and 3 copies per reaction. The platform was confirmed for detecting multiple E. coli strains, and for water samples of different salt concentrations. We validated the functions of the platform by analyzing recreational water samples collected near the Atlantic Ocean that contain different concentrations of salt and bacteria., (© 2023. The Author(s).)

Bacterial biofilms are surface-attached communities of slow- or non-replicating cells embedded within a protective matrix of biomolecules. Unlike free-floating planktonic bacteria, biofilms are innately tolerant to conventional antibiotics and are prevalent in recurring and chronic infections. Nitroxoline, a broad-spectrum biofilm-eradicating agent, was used to probe biofilm viability. Transcript profiling (RNA-seq) showed that 452 of 2594 genes (17.4%) in methicillin-resistant Staphylococcus aureus (MRSA) biofilms were differentially expressed after a 2 h treatment of nitroxoline. WoPPER analysis and time-course validation (RT-qPCR) revealed that gene clusters involved in iron acquisition ( sbn , isd , MW2101, MW0695, fhu , and feo ) were rapidly up-regulated following nitroxoline treatment, which is indicative of iron starvation in MRSA biofilms. In addition, genes related to oligopeptide transporters and riboflavin biosynthesis were found to be up-regulated, while genes related to carotenoid biosynthesis and nitrate assimilation were down-regulated. RT-qPCR experiments revealed that iron uptake transcripts were also up-regulated in established Staphylococcus epidermidis and Acinetobacter baumannii biofilms following nitroxoline treatment. Overall, we show RNA-seq to be an ideal platform to define cellular pathways critical for biofilm survival, in addition to demonstrating the need these bacterial communities have for iron.

During infection, bacteria use an arsenal of resistance mechanisms to negate antibiotic therapies. In addition, pathogenic bacteria form surface-attached biofilms bearing enriched populations of metabolically dormant persister cells. Bacteria develop resistance in response to antibiotic insults; however, nonreplicating biofilms are innately tolerant to all classes of antibiotics. As such, molecules that can eradicate antibiotic-resistant and antibiotic-tolerant bacteria are of importance. Here, we report modular synthetic routes to fluorine-containing halogenated phenazine (HP) and halogenated acridine (HA) agents with potent antibacterial and biofilm-killing activities. Nine fluorinated phenazines were rapidly accessed through a synthetic strategy involving (1) oxidation of fluorinated anilines to azobenzene intermediates, (2) S N Ar with 2-methoxyaniline, and (3) cyclization to phenazines upon treatment with trifluoroacetic acid. Five structurally related acridine heterocycles were synthesized using S N Ar and Buchwald-Hartwig approaches. From this focused collection, phenazines 5g , 5h , 5i , and acridine 9c demonstrated potent antibacterial activities against Gram-positive pathogens (MIC = 0.04-0.78 μM). Additionally, 5g and 9c eradicated Staphylococcus aureus , Staphylococcus epidermidis and Enterococcus faecalis biofilms with excellent potency ( 5g , MBEC = 4.69-6.25 μM; 9c , MBEC = 4.69-50 μM). Using real-time quantitative polymerase chain reaction (RT-qPCR), 5g , 5h , 5i , and 9c rapidly induce the transcription of iron uptake biomarkers isdB and sbnC in methicillin-resistant S. aureus (MRSA) biofilms, and we conclude that these agents operate through iron starvation. Overall, fluorinated phenazine and acridine agents could lead to ground-breaking advances in the treatment of challenging bacterial infections.

Antibiotic-resistant infections present significant challenges to patients. As a result, there is considerable need for new antibacterial therapies that eradicate pathogenic bacteria through non-conventional mechanisms. Our group has identified a series of halogenated phenazine (HP) agents that induce rapid iron starvation that leads to potent killing of methicillin-resistant Staphylococcus aureus biofilms. Here, we report the design, chemical synthesis and microbiological assessment of a HP-quinone ether prodrug model aimed to (1) eliminate general (off-target) iron chelation, and (2) release an active HP agent through the bioreduction of a quinone trigger. Here, we demonstrate prodrug analogue HP-29-Q to have a stable ether linkage that enables HP release and moderate to good antibacterial activities against lab strains and multi-drug resistant clinical isolates.

Pathogenic bacteria demonstrate incredible abilities to evade conventional antibiotics through the development of resistance and formation of dormant, surface-attached biofilms. Therefore, agents that target and eradicate planktonic and biofilm bacteria are of significant interest. We explored a new series of halogenated phenazines (HP) through the use of N -aryl-2-nitrosoaniline synthetic intermediates that enabled functionalization of the 3-position of this scaffold. Several HPs demonstrated potent antibacterial and biofilm-killing activities ( e.g. , HP 29 , against methicillin-resistant Staphylococcus aureus : MIC = 0.075 μM; MBEC = 2.35 μM), and transcriptional analysis revealed that HPs 3 , 28 , and 29 induce rapid iron starvation in MRSA biofilms. Several HPs demonstrated excellent activities against Mycobacterium tuberculosis (HP 34 , MIC = 0.80 μM against CDC1551). This work established new SAR insights, and HP 29 demonstrated efficacy in dorsal wound infection models in mice. Encouraged by these findings, we believe that HPs could lead to significant advances in the treatment of challenging infections.

There is a significant need for new antibacterial agents as pathogenic bacteria continue to threaten human health through the acquisition of resistance and tolerance towards existing antibiotics. Over the last several years, our group has been developing a novel series of halogenated phenazines that demonstrate potent antibacterial and biofilm eradication activities against critical Gram-positive pathogens, including: Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecium. Here, we report the design, chemical synthesis and initial biological assessment of a halogenated phenazine-erythromycin conjugate prodrug 5 aimed at enhancing the translational potential for halogenated phenazines as a treatment of bacterial infections.

During infection of a host, Pseudomonas aeruginosa orchestrates global gene expression to adapt to the host environment and counter the immune attacks. P. aeruginosa harbours hundreds of regulatory genes that play essential roles in controlling gene expression. However, their contributions to the bacterial pathogenesis remain largely unknown. In this study, we analysed the transcriptomic profile of P. aeruginosa cells isolated from lungs of infected mice and examined the roles of upregulated regulatory genes in bacterial virulence. Mutation of a novel regulatory gene pvrA (PA2957) attenuated the bacterial virulence in an acute pneumonia model. Chromatin immunoprecipitation (ChIP)-Seq and genetic analyses revealed that PvrA directly regulates genes involved in phosphatidylcholine utilization and fatty acid catabolism. Mutation of the pvrA resulted in defective bacterial growth when phosphatidylcholine or palmitic acid was used as the sole carbon source. We further demonstrated that palmitoyl coenzyme A is a ligand for the PvrA, enhancing the binding affinity of PvrA to its target promoters. An arginine residue at position 136 was found to be essential for PvrA to bind palmitoyl coenzyme A. Overall, our results revealed a novel regulatory pathway that controls genes involved in phosphatidylcholine and fatty acid utilization and contributes to the bacterial virulence., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objectives: A group of ST664 XDR Pseudomonas aeruginosa strains have been isolated from a burn clinic. Here we decipher their resistomes and likely mechanisms of resistance acquisition., Methods: The complete nucleotide sequences of representative isolates were determined, by PacBio and Illumina MiSeq sequencing, and analysed for antimicrobial resistance (AMR) genes as well as sequence variations. S1-PFGE was used to determine the sizes and numbers of plasmids harboured by the isolates. Purified plasmid DNA was further sequenced by PacBio technology, closed manually and annotated by RAST. The mobility of plasmids was determined by conjugation assays., Results: The XDR P. aeruginosa ST664 clone carries 11 AMR genes, including a blaKPC-2 gene that confers resistance to carbapenems. Most of the ST664 isolates carry three coexisting plasmids. blaKPC-2 and a cluster of three AMR genes (aadB-cmlA1-sul1) are encoded on a 475 kb megaplasmid pNK546a, which codes for an IncP-3-like replication and partitioning mechanism, but has lost the conjugative transfer system. Interestingly, however, pNK546a is mobilizable and can be transferred to P. aeruginosa PAO1 with the help of a co-residing IncP-7 conjugative plasmid. The blaKPC-2 gene is carried by an IS6100-ISKpn27-blaKPC-2-ΔISKpn6-Tn1403 mobile element, which might be brought into the ST664 clone by another co-resident IncP-1α plasmid, which is inclined to be lost. Moreover, pNK546a harbours multiple heavy metal (mercury, tellurite and silver) resistance modules., Conclusions: To the best of our knowledge, pNK546a is the first fully sequenced blaKPC-2-carrying megaplasmid from P. aeruginosa. These results give new insights into bacterial adaptation and evolution during nosocomial infections., (© The Author(s) 2020. 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.)

Intracellular delivery of functional proteins is of great interest for basic biological research as well as for clinical applications. Transfection is the most commonly used method, however, it is not applicable to large-scale manipulation and inefficient in important cell types implicated in biomedical applications, such as epithelial, immune and pluripotent stem cells. In this study, we explored a bacterial type III secretion system (Bac-T3SS)-mediated proteofection method to overcome these limitations. An attenuated Pseudomonas aeruginosa vector was constructed, which has features of low toxicity, high T3SS activity, and self-limiting growth. Compared to the method of transfection, the Bac-T3SS showed significantly higher efficiencies of Cre recombinase translocation and target site recombination for hard-to-transfect human cell lines. Furthermore, through the delivery of β-lactamase in live animals, we demonstrated the feasibility and biosafety of in vivo application of the Bac-T3SS. This study provided an efficient and low-cost proteofection strategy for laboratory use as well as for application in large-scale cell manipulations., (© 2019 Wiley Periodicals, Inc.)

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.)

IL-1β expression is increased in response to P. aeruginosa infection, but the responsible proteins have not been clearly elucidated. Here, we demonstrate for the first time that IL-1β expression is induced in response to the heat shock protein 70-like protein DnaK. Treatment with recombinant DnaK (rDnaK) increased IL-1β expression in a dose- and time-dependent manner, and the release of mature IL-1β in response to rDnaK was detected to an extent similar to that stimulated by the well-known agonists, lipopolysaccharide and nigericin. rDnaK-mediated IL-1β expression was driven by the NF-κB signaling pathway. In addition, expression was controlled by the JNK signaling pathway, although these two signaling cascades act independently upon rDnaK stimulation. Finally, rDnaK-induced IL-1β expression was initiated via the action of TLR4. Taken together, the data reveal that P. aeruginosa-derived DnaK induces expression of IL-1β via TLR4-dependent activation of the NF-κB and JNK signaling pathways., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Pseudomonas aeruginosa is an opportunistic pathogenic bacterium whose type III secretion system (T3SS) plays a critical role in acute infections. Translocation of the T3SS effectors into host cells induces cytotoxicity. In addition, the T3SS promotes the intracellular growth of P. aeruginosa during host infections. The T3SS regulon genes are regulated by an AraC-type regulator, ExsA. In this study, we found that an extracellular metalloprotease encoded by impA (PA0572) is under the regulation of ExsA. An ExsA consensus binding sequence was identified upstream of the impA gene, and direct binding of the site by ExsA was demonstrated via an electrophoretic mobility shift assay. We further demonstrate that secreted ImpA cleaves the macrophage surface protein CD44, which inhibits the phagocytosis of the bacterial cells by macrophages. Combined, our results reveal a novel ExsA-regulated virulence factor that cooperatively inhibits the functions of macrophages with the T3SS., (Copyright © 2019 American Society for Microbiology.)

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.)

The biofilm formation by Pseudomonas aeruginosa highly increases the bacterial resistance to antimicrobial agents and host immune clearance. The biofilm formation is positively regulated by two small RNAs, RsmY and RsmZ. Previously, we reported that mutation in the polynucleotide phosphorylase (PNPase) coding gene pnp increases the levels of RsmY/Z. However, in this study, we found that the biofilm formation is decreased in the pnp mutant, which is due to a defect in rhamnolipids production. The rhamnolipids production is regulated by the RhlI-RhlR quorum sensing system. We found that PNPase influences the translation of RhlI through its 5'-untranslated region (UTR) and identified that the sRNA P27 is responsible for the translational repression. In vitro translation experiments demonstrated that P27 directly represses the translation of the rhlI mRNA through its 5'UTR in an Hfq-dependent manner. Point mutations in the rhlI 5'UTR or P27, which abolish the pairing between the two RNAs restore the rhlI expression and rhamnolipids production as well as the biofilm formation in the pnp mutant. Overall, our results reveal a novel layer of regulation of the Rhl quorum sensing system by the sRNA P27., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes various acute and chronic infections. It is intrinsically resistant to a variety of antibiotics. However, production of pyocins during SOS response sensitizes P. aeruginosa to quinolone antibiotics by inducing cell lysis. The polynucleotide phosphorylase (PNPase) is a conserved phosphate-dependent 3'-5' exonuclease that plays an important role in bacterial response to environmental stresses and pathogenesis by influencing mRNA and small RNA stabilities. Previously, we demonstrated that PNPase controls the type III and type VI secretion systems in P. aeruginosa . In this study, we found that mutation of the PNPase coding gene ( pnp ) increases the bacterial resistance to ciprofloxacin. Gene expression analyses revealed that the expression of pyocin biosynthesis genes is decreased in the pnp mutant. PrtR, a negative regulator of pyocin biosynthesis genes, is upregulated in the pnp mutant. We further demonstrated that PNPase represses the expression of PrtR on the post-transcriptional level. A fragment containing 43 nucleotides of the 5' untranslated region was found to be involved in the PNPase mediated regulation of PrtR. Overall, our results reveled a novel layer of regulation on the pyocin biosynthesis by the PNPase in P. aeruginosa .

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.)

The Pseudomonas aeruginosa RsaL is a negative regulator of the quorum sensing signal synthesis gene lasI. The expression of RsaL is directly activated by the LasI cognate regulator LasR. Thus, RsaL and LasI-LasR (LasI/R) form a regulatory loop. Further studies revealed that RsaL is a global regulator which controls the expression of numerous genes through quorum sensing system dependent and independent pathways. However, whether RsaL is involved in antibiotic tolerance remains elusive. In this study, we found that the mutation of rsaL increased bacterial tolerance to ciprofloxacin and carbenicillin. Through motif search, gene expression analyses and electrophoretic mobility shift assays, we found that RsaL directly represses the expression of the narK1K2GHJI operon, which is involved in the tolerance to ciprofloxacin. We further demonstrated that the narK1K2GHJI operon is directly regulated by LasR. In combination, our study revealed a novel operon under the control of the RsaL, LasI/R regulatory loop.

The type III secretion system (T3SS) plays an important role in the pathogenesis of Pseudomonas aeruginosa . Expression of the T3SS is controlled under a complicate regulatory network. In this study, we demonstrate that NrtR (PA4916) is involved in the T3SS expression and pathogenesis of P. aeruginosa in a mouse acute pneumonia model. Overexpression of the T3SS central activator ExsA or exogenous supplementation of cAMP restored the expression of T3SS in the Δ nrtR mutant, suggesting that NrtR might regulate T3SS through the cAMP-Vfr signaling pathway. Further experiments demonstrated that the decrease of cAMP content is not due to the expression change of adenylate cyclases or phosphodiesterase in the Δ nrtR mutant. As it has been shown that nadD2 is upregulated in the Δ nrtR mutant, we overexpressed nadD2 in wild type PAK, which reduced the intracellular cAMP level and the expression of the T3SS genes. Meanwhile, deletion of nadD2 in the Δ nrtR mutant restored the expression and secretion of the T3SS. Co-immunoprecipitation assay revealed an interaction between NadD2 and the catalytic domain of the adenylate cyclase CyaB. Further in vitro assay indicated that NadD2 repressed the enzymatic activity of CyaB. Therefore, we have identified a novel regulatory mechanism of T3SS in P. aeruginosa .

Bacterial biofilms are surface-attached communities of non-replicating bacteria innately tolerant to antibiotics. Biofilms display differential gene expression profiles and physiologies as compared to their planktonic counterparts; however, their biology remains largely unknown. In this study, we used a halogenated phenazine (HP) biofilm eradicator in transcript profiling experiments (RNA-seq) to define cellular targets and pathways critical to biofilm viability. WoPPER analysis with time-course validation (RT-qPCR) revealed that HP-14 induces rapid iron starvation in MRSA biofilms, as evident by the activation of iron-acquisition gene clusters in 1 hour. Serine proteases and oligopeptide transporters were also found to be up-regulated, whereas glycolysis, arginine deiminase, and urease gene clusters were down-regulated. KEGG analysis revealed that HP-14 impacts metabolic and ABC transporter functional pathways. These findings suggest that MRSA biofilm viability relies on iron homeostasis., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Toxin-antitoxin (TA) systems play important roles in bacteria persister formation. Increasing evidence demonstrate the roles of TA systems in regulating virulence factors in pathogenic bacteria. The toxin HigB in Pseudomonas aeruginosa contributes to persister formation and regulates the expression of multiple virulence factors and biofilm formation. However, the regulatory mechanism remains elusive. In this study, we explored the HigB mediated regulatory pathways. We demonstrate that HigB decreases the intracellular level of c-di-GMP, which is responsible for the increased expression of the type III secretion system (T3SS) genes and repression of biofilm formation. By analyzing the expression levels of the known c-di-GMP metabolism genes, we find that three c-di-GMP hydrolysis genes are up regulated by HigB, namely PA2133, PA2200 and PA3825. Deletion of the three genes individually or simultaneously diminishes the HigB mediated regulation on the expression of T3SS genes and biofilm formation. Therefore, our results reveal novel functions of HigB in P. aeruginosa .

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 .

Antibiotic-resistant bacteria and surface-attached biofilms continue to play a significant role in human health and disease. Innovative strategies are needed to identify new therapeutic leads to tackle infections of drug-resistant and tolerant bacteria. We synthesized a focused library of 14 new halogenated quinolines to investigate the impact of ClogP values on antibacterial and biofilm-eradication activities. During these investigations, we found select polar appendages at the 2-position of the HQ scaffold were more well-tolerated than others. We were delighted to see multiple compounds display enhanced activities against the major human pathogen S. epidermidis. In particular, HQ 2 (ClogP = 3.44) demonstrated enhanced activities against MRSE 35984 planktonic cells (MIC = 0.59 μM) compared to MRSA and VRE strains in addition to potent MRSE biofilm eradication activities (MBEC = 2.35 μM). Several of the halogenated quinolines identified here reported low cytotoxicity against HeLa cells with minimal hemolytic activity against red blood cells. We believe that halogenated quinoline small molecules could play an important role in the development of next-generation antibacterial therapeutics capable of targeting and eradicating biofilm-associated infections., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Bacterial biofilms are surface-attached communities comprised of nonreplicating persister cells housed within a protective extracellular matrix. Biofilms display tolerance toward conventional antibiotics, occur in ∼80% of infections, and lead to >500000 deaths annually. We recently identified halogenated phenazine (HP) analogues which demonstrate biofilm-eradicating activities against priority pathogens; however, the synthesis of phenazines presents limitations. Herein, we report a refined HP synthesis which expedited the identification of improved biofilm-eradicating agents. 1-Methoxyphenazine scaffolds were generated through a Buchwald-Hartwig cross-coupling (70% average yield) and subsequent reductive cyclization (68% average yield), expediting the discovery of potent biofilm-eradicating HPs (e.g., 61: MRSA BAA-1707 MBEC = 4.69 μM). We also developed bacterial-selective prodrugs (reductively activated quinone-alkyloxycarbonyloxymethyl moiety) to afford HP 87, which demonstrated excellent antibacterial and biofilm eradication activities against MRSA BAA-1707 (MIC = 0.15 μM, MBEC = 12.5 μM). Furthermore, active HPs herein exhibit negligible cytotoxic or hemolytic effects, highlighting their potential to target biofilms.

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.

Virus-mediated expression of defined transcription factor (TF) genes can effectively induce cellular reprogramming. However, sustained expression of the TFs often hinders pluripotent stem cell (PSC) differentiation into specific cell types, as each TF exerts its effect on PSCs for a defined period of time during differentiation. Here, we applied a bacterial type III secretion system (T3SS)-based protein delivery tool to directly translocate TFs in the form of protein into human PSCs. This transient protein delivery technique showed high delivery efficiency for hPSCs, and it avoids potential genetic alterations caused by the introduction of transgenes. In an established cardiomyocyte de novo differentiation procedure, five transcriptional factors, namely GATA4, MEF2C, TBX5, ESRRG and MESP1 (abbreviated as GMTEM), were translocated at various time points. By detecting the expression of cardiac marker genes (Nkx2.5 and cTnT), we found that GMTEM proteins delivered on mesoderm stage of the cardiomyocytes lineage differentiation significantly enhanced both the human ESC and iPSC differentiation into cardiomyocytes, while earlier or later delivery diminished the enhancing effect. Furthermore, all of the five factors were required to enhance the cardiac differentiation. This work provides a virus-free strategy of transient transcription factors delivery for directing human stem cell fate without jeopardizing genome integrity, thus safe for biomedical applications.

Pseudomonas aeruginosa , a Gram-negative opportunistic pathogenic bacterium, causes acute and chronic infections. Upon entering the host, P. aeruginosa alters global gene expression to adapt to host environment and avoid clearance by the host immune system. Proline utilization A (PutA) is a bifunctional enzyme, which converts proline to glutamate. Here we report that PutA was required for the virulence of P. aeruginosa in a murine acute pneumonia model. A putA mutant was more susceptible to oxidative stress compared to the wild type strain. An AraC/XylS family protein, PruR, directly bound to the upstream of -35 box in the putA promoter and activated putA expression. High concentration of proline in bacteria up-regulated pruR expression, which led to the activation of putA expression. As a feedback regulation, glutamate produced by PutA released PruR from the putA promoter and turned off the putA expression. PruR affected bacterial virulence through the regulation of the putA expression. Altogether, these data are the first to reveal that PutA plays an important role in the pathogenesis of P. aeruginosa , as well as to describe the genetic regulation of PutA in P. aeruginosa .

A protein delivery tool based on bacterial type III secretion system (T3SS) has been broadly applied in biomedical researches. In this review, we summarize various applications of the T3SS-mediate protein delivery which enables translocation of proteins directly into mammalian cells without protein purification. Some of the remarkable advancements include delivery of antigens for therapeutic vaccines, nucleases for genome editing, transcription factors for cellular reprogramming and stem cells differentiation, and signaling molecules for post-translational proteomics studies. With continued improvement of the T3SS-mediated protein delivery tools, even wider application of the technology is anticipated., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.)

Pseudomonas aeruginosa is an opportunistic human pathogen that is prevalent in hospitals and continues to develop resistance to multiple classes of antibiotics. Historically, β-lactam antibiotics have been the first line of therapeutic defense. However, the emergence of multidrug-resistant (MDR) strains of P. aeruginosa, such as AmpC β-lactamase overproducing mutants, limits the effectiveness of current antibiotics. Among AmpC hyperproducing clinical isolates, inactivation of AmpG, which is essential for the expression of AmpC, increases bacterial sensitivity to β-lactam antibiotics. We hypothesize that inhibition of AmpG activity will enhance the efficacy of β-lactams against P. aeruginosa. Here, using a highly drug-resistant AmpC-inducible laboratory strain PAO1, we describe an ultra-high-throughput whole-cell turbidity assay designed to identify small-molecule inhibitors of the AmpG. We screened 645,000 compounds to identify compounds with the ability to inhibit bacterial growth in the presence of cefoxitin, an AmpC inducer, and identified 2663 inhibitors that were also tested in the absence of cefoxitin to determine AmpG specificity. The Z' and signal-to-background ratio were robust at 0.87 ± 0.05 and 2.2 ± 0.2, respectively. Through a series of secondary and tertiary studies, including a novel luciferase-based counterscreen, we ultimately identified eight potential AmpG-specific inhibitors.

Exoenzyme Y (ExoY) is a type III secretion system effector found in 90% of the Pseudomonas aeruginosa isolates. Although it is known that ExoY is a soluble nucleotidyl cyclase that increases the cytoplasmic levels of nucleoside 3',5'-cyclic monophosphates (cNMPs) to mediate endothelial Tau phosphorylation and permeability, its functional role in the innate immune response is still poorly understood. Transforming growth factor β-activated kinase 1 (TAK1) is critical for mediating Toll-like receptor (TLR) signaling and subsequent activation of NF-κB and AP-1, which are transcriptional activators of innate immunity. Here, we report that ExoY inhibits proinflammatory cytokine production through suppressing the activation of TAK1 as well as downstream NF-κB and mitogen-activated protein (MAP) kinases. Mice infected with ExoY-deficient P. aeruginosa had higher levels of tumor necrosis factor (TNF) and interleukin-6 (IL-6), more neutrophil recruitment, and a lower bacterial load in lung tissue than mice infected with wild-type P. aeruginosa Taken together, our findings identify a previously unknown mechanism by which P. aeruginosa ExoY inhibits the host innate immune response., (Copyright © 2017 American Society for Microbiology.)

Many Pseudomonas aeruginosa virulence traits that contribute to human infections are accepted as being associated with its environmental lifestyle. Therefore, identifying the molecular mechanisms that govern the lifestyle choice is of high significance. We previously reported that a mutation in suhB results in a decrease in swimming motility and increased biofilm formation compared to the wild-type strain. Yet, little is known about how this occurs. In this study, we demonstrated that SuhB inversely regulates motility and biofilm formation through the GacA-RsmY/Z-RsmA cascade. Mutations in gacA or the two small RNAs rsmY / rsmZ , or overproduction of the RsmA protein essentially rescued the motility defect of the suhB mutant. Additionally, we identified a c-di-GMP mediated mechanism for SuhB regulation of motility and biofilm formation. We showed that the Δ suhB mutant displayed elevated levels of c-di-GMP, and the Δ suhB motility and biofilm phenotypes could be switched by artificially decreasing c-di-GMP levels. Further experiments led to the identification of the diguanylate cyclase GcbA responsible for regulating the c-di-GMP concentration in Δ suhB and hence the switch between planktonic and surface-associated growth. Together, our results demonstrate a novel mechanism for SuhB regulation of the lifestyle transition via the Gac/Rsm and c-di-GMP signaling networks in P. aeruginosa .

Unlike individual, free-floating planktonic bacteria, biofilms are surface-attached communities of slow- or non-replicating bacteria encased within a protective extracellular polymeric matrix enabling persistent bacterial populations to tolerate high concentrations of antimicrobials. Our current antibacterial arsenal is composed of growth-inhibiting agents that target rapidly-dividing planktonic bacteria but not metabolically dormant biofilm cells. We report the first modular synthesis of a library of 20 halogenated phenazines (HP), utilizing the Wohl-Aue reaction, that targets both planktonic and biofilm cells. New HPs, including 6-substituted analogues, demonstrate potent antibacterial activities against MRSA, MRSE and VRE (MIC = 0.003-0.78 µM). HPs bind metal(II) cations and demonstrate interesting activity profiles when co-treated in a panel of metal(II) cations in MIC assays. HP 1 inhibited RNA and protein biosynthesis while not inhibiting DNA biosynthesis using 3 H-radiolabeled precursors in macromolecular synthesis inhibition assays against MRSA. New HPs reported here demonstrate potent eradication activities (MBEC = 0.59-9.38 µM) against MRSA, MRSE and VRE biofilms while showing minimal red blood cell lysis or cytotoxicity against HeLa cells. PEG-carbonate HPs 24 and 25 were found to have potent antibacterial activities with significantly improved water solubility. HP small molecules could have a dramatic impact on persistent, biofilm-associated bacterial infection treatments.

Fis is a versatile DNA binding protein in bacteria. It has been demonstrated in multiple bacteria that Fis plays crucial roles in regulating bacterial virulence factors and optimizing bacterial adaptation to various environments. However, the role of Fis in Pseudomonas aeruginosa virulence as well as gene regulation remains largely unknown. Here, we found that Fis was required for the virulence of P. aeruginosa in a murine acute pneumonia model. Transcriptome analysis revealed that expression of T3SS genes, including master regulator ExsA, was defective in a fis ::Tn mutant. We further demonstrate that the continuous transcription of exsC, exsE, exsB , and exsA driven by the exsC promoter was required for the activation of T3SS. Fis was found to specifically bind to the exsB - exsA intergenic region and plays an essential role in the transcription elongation from exsB to exsA . Therefore, we found a novel role of Fis in the regulation of exsA expression.

During host infection, Pseudomonas aeruginosa coordinately regulates the expression of numerous genes to adapt to the host environment while counteracting host clearance mechanisms. As infected patients take antibiotics, the invading bacteria encounter antibiotics in the host milieu. P. aeruginosa is highly resistant to antibiotics due to multiple chromosomally encoded resistant determinants. And numerous in vitro studies have demonstrated the regulatory mechanisms of antibiotic resistance related genes in response to antibiotics. However, it is not well-known how host environment affects bacterial response to antibiotics. In this study, we found that P. aeruginosa cells directly isolated from mice lungs displayed higher susceptibility to tobramycin than in vitro cultured bacteria. In vitro experiments demonstrated that incubation with A549 and differentiated HL60 (dHL60) cells sensitized P. aeruginosa to tobramycin. Further studies revealed that reactive oxygen species produced by the host cells contributed to the increased bacterial susceptibility. At the same concentration of tobramycin, presence of A549 and dHL60 cells resulted in higher expression of heat shock proteins, which are known inducible by tobramycin. Further analyses revealed decreased membrane potential upon incubation with the host cells and modification of lipopolysaccharide, which contributed to the increased susceptibility to tobramycin. Therefore, our results demonstrate that contact with host cells increased bacterial susceptibility to tobramycin.

Bacterial oligoribonuclease (Orn) is a conserved 3'-to-5' exonuclease. In Pseudomonas aeruginosa , it has been demonstrated that Orn plays a major role in the hydrolysis of pGpG, which is required for cyclic-di-GMP homeostasis. Meanwhile, Orn is involved in the degradation of nanoRNAs, which can alter global gene expression by serving as transcription initiation primers. Previously, we found that Orn is required for the type III secretion system and pathogenesis of P. aeruginosa , indicating a role of Orn in the bacterial response to environmental stimuli. Here we report that Orn is required for the tolerance of P. aeruginosa to ciprofloxacin. Transcriptome analysis of an orn mutant revealed the upregulation of pyocin biosynthesis genes. Mutation of genes involved in pyocin biosynthesis in the background of an orn mutant restored bacterial tolerance to ciprofloxacin. We further demonstrate that the upregulation of pyocin biosynthesis genes is due to RecA-mediated autoproteolysis of PrtR, which is the major negative regulator of pyocin biosynthesis genes. In addition, the SOS response genes were upregulated in the orn mutant, indicating a DNA damage stress. Therefore, our results revealed a novel role of Orn in bacterial tolerance to ciprofloxacin., (Copyright © 2017 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.)

Pseudomonas aeruginosa is a Gram negative opportunistic pathogenic bacterium, which causes acute and chronic infections. Upon entering the host, bacteria alter global gene expression to adapt to host environment and avoid clearance by the host. Enolase is a glycolytic enzyme involved in carbon metabolism. It is also a component of RNA degradosome, which is involved in RNA processing and gene regulation. Here, we report that enolase is required for the virulence of P. aeruginosa in a murine acute pneumonia model. Mutation of enolase coding gene ( eno ) increased bacterial susceptibility to neutrophil mediated killing, which is due to reduced tolerance to oxidative stress. Catalases and alkyl hydroperoxide reductases play a major role in protecting the cell from oxidative damages. In the eno mutant, the expression levels of catalases (KatA and KatB) were similar as those in the wild type strain in the presence of H 2 O 2 , however, the expression levels of alkyl hydroperoxide reductases (AhpB and AhpC) were significantly reduced. Overexpression of ahpB but not ahpC in the eno mutant fully restored the bacterial resistance to H 2 O 2 as well as neutrophil mediated killing, and partially restored bacterial virulence in the murine acute pneumonia model. Therefore, we have identified a novel role of enolase in the virulence of P. aeruginosa .

AmpG is a transmembrane protein with permease activity that transports meuropeptide from the periplasm to the cytoplasm, which is essential for the induction of the ampC encoding β-lactamase. To obtain new insights into the relationship between AmpG structure and function, comparative genomics analysis, secondary and tertiary structure modeling, site-directed mutational analyses and genetic complementation experiments were performed in this study. AmpGs from different genera of bacteria (Escherichia coli, Vibrio cholerae and Acinetobacter baumannii) could complement AmpG function in Pseudomonas aeruginosa. The minimal inhibitory concentration (MIC) to ampicillin is 512 μg/ml for wild type strain PAO1, while it is 32 μg/ml for an ampG deletion mutant strain (PAO1ΔampG) with a corresponding decrease in the activity of the ampC-encoded β-lactamase. Site-directed mutagenesis of conserved AmpG residues (G29, A129, Q131 and A197) resulted in a loss of function, resulting in a loss of resistance to ampicillin in PAO1ΔampG. The G29A, G29V, A129T, A129V, A129D, A197S and A197D mutants had lower resistance to ampicillin and significantly decreased activity of the AmpC β-lactamase. The G29A, G29V, A129V, A197S and A197D mutants had decreased ampG mRNA transcript levels. The A129T and A129D mutants had normal ampG mRNA transcript levels, but the function of the protein was drastically reduced. Our experimental results demonstrate that the conserved amino acids played essential roles in maintaining the function of AmpG. Combined with the AmpG structural information, these critical amino acids can be targeted for the development of new anti-bacterial agents., Competing Interests: The authors have declared that no competing interests exist.

Acinetobacter baumannii is a leading pathogen responsible for nosocomial infections. The emergence of carbapenem-resistant A. baumannii (CRAB) has left few effective antibiotics for clinicians to use. To investigate the temporal evolutionary relationships among CRAB strains, we collected 248 CRAB isolates from a Chinese burns institute over 3 years. The prevalence of the OXA-23 gene was detected by polymerase chain reaction. Multilocus sequence typing was used to type the CRAB strains and eBURST was used to analyze their evolutionary relationships. Wound surfaces (41%), sputa (24%), catheters (15%), and bloods (14%) were the four dominant isolation sources. Except for minocycline (33.5%) and sulbactam/cefoperazone (74.6%), these CRAB strains showed high resistance rates (>90%) to 16 tested antibiotics. The 248 isolates fall into 26 sequence types (STs), including nine known STs and 17 unknown STs. The majority (230/248) of these isolates belong to clonal complex 92 (CC92), including eight isolates belonging to seven unreported STs. A new CC containing 11 isolates grouped into four new STs was identified. The OXA-23 gene was detected at high prevalence among the CRAB isolates and the prevalence rate among the various STs differed. The majority of the isolates displayed a close evolutionary relationship, suggesting that serious nosocomial spreading and nosocomial infections of CRAB have occurred in the burn unit. In conclusion, the main CC for CRAB in this Chinese burn unit remained unchanged during the 3-year study period, and a new CC was identified. CC92 was the dominant complex, and more attention should be directed toward monitoring the new CC we have identified herein.

Acinetobacter baumannii infection is a serious threat to burn patients. Bacteremia due to A. baumannii is becoming the most common cause of mortality following burn. However, the epidemiology of A. baumannii causing burn-related bloodstream infections has rarely been reported. We retrospectively collected 81 A. baumannii isolates from the bloodstream of burn patients over a three-year period. Antibiotic susceptibility tests, the prevalence of antibiotic-resistant genes and sequence typing (ST) were conducted to characterize these strains. Most of the isolates showed an extensive drug-resistant phenotype. The resistance frequencies to imipenem and meropenem were 94% and 91%, respectively. The blaOXA-23-like gene, AmpC, IS-AmpC, PER and SIM are the five most prevalent resistant genes, and their prevalence rates are 93% (75/81), 86% (70/81), 73% (59/81), 73% (59/81) and 52% (42/81), respectively. The 81 isolates were grouped into 10 known and 18 unknown ST types, with ST368 (38%) being the most prevalent. Except for ST457 and four new types (STn2, STn6, STn11 and STn14), the remaining 23 ST types belonged to one clonal complex 92, which is most common among clinical isolate in China. The above results indicated that ST368 isolates possessing both the blaOXA-23-like gene and ampC gene were the main culprits of the increasing nosocomial A. baumannii infection in this study. More attention should be paid to monitoring the molecular epidemiology of A. baumannii isolates from burn patients to prevent further distribution. Such information may help clinicians with therapeutic decisions and infection control in the Burns Institute., (Copyright © 2016. Published by Elsevier Ltd.)

Bacterial persister cells are dormant and highly tolerant to lethal antibiotics, which are believed to be the major cause of recurring and chronic infections. Activation of toxins of bacterial toxin-antitoxin systems inhibits bacterial growth and plays an important role in persister formation. However, little is known about the overall gene expression profile upon toxin activation. More importantly, how the dormant bacterial persisters evade host immune clearance remains poorly understood. Here we demonstrate that a Pseudomonas aeruginosa toxin-antitoxin system HigB-HigA is required for the ciprofloxacin induced persister formation. Transcriptome analysis of a higA ::Tn mutant revealed up regulation of type III secretion systems (T3SS) genes. Overexpression of HigB increased the expression of T3SS genes as well as bacterial cytotoxicity. We further demonstrate that wild type bacteria that survived ciprofloxacin treatment contain higher levels of T3SS proteins and display increased cytotoxicity to macrophage compared to vegetative bacterial cells. These results suggest that P. aeruginosa accumulates T3SS proteins during persister formation, which can protect the persister cells from host clearance by efficiently killing host immune cells.

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.)

Oligoribonuclease (Orn) is a 3' to 5' exonuclease that degrades nanoRNAs, which can serve as primers for transcription initiation at a significant fraction of promoters. One of Orn's substrates, pGpG inhibits the enzymatic activity of EAL-domain containing phosphodiesterases (PDEs), thereby increasing intracellular cyclic-di-GMP (c-di-GMP) level. Here, we found that an orn mutant of Pseudomonas aeruginosa displayed reduced cytotoxicity, which was mainly due to deficient type III secretion system (T3SS). Given the importance of T3SS in pathogenicity, we examined the bacterial virulence in a mouse acute pneumonia model and found that the Δorn mutant was highly attenuated compared to the wild type PA14 strain. Overexpression of an EAL domain-containing PDE reduced the c-di-GMP level as well as biofilm formation in the Δorn mutant. However, no effect was observed on the expression of T3SS genes, suggesting that increased c-di-GMP level is not the solely cause of defective T3SS in the Δorn mutant. Overall, our results demonstrated an essential role of Orn in the expression of T3SS as well as pathogenesis of P. aeruginosa., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Agents capable of eradicating bacterial biofilms are of great importance to human health as biofilm-associated infections are tolerant to our current antibiotic therapies. We have recently discovered that halogenated quinoline (HQ) small molecules are: 1) capable of eradicating methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE) and vancomycin-resistant Enterococcus faecium (VRE) biofilms, and 2) synthetic tuning of the 2-position of the HQ scaffold has a significant impact on antibacterial and antibiofilm activities. Here, we report the chemical synthesis and biological evaluation of 39 HQ analogues that have a high degree of structural diversity at the 2-position. We identified diverse analogues that are alkylated and aminated at the 2-position of the HQ scaffold and demonstrate potent antibacterial (MIC≤0.39 μm) and biofilm eradication (MBEC 1.0-93.8 μm) activities against drug-resistant Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecium strains while demonstrating <5 % haemolysis activity against human red blood cells (RBCs) at 200 μm. In addition, these HQs demonstrated low cytotoxicity against HeLa cells. Halogenated quinolines are a promising class of antibiofilm agents against Gram-positive pathogens that could lead to useful treatments against persistent bacterial infections., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Expression of a putative acyltransferase encoded by NCgl- 0350 of Corynebacterium glutamicum is induced by cell-free culture fluids obtained from stationary-phase growth of both C. glutamicum and Pseudomonas aeruginosa, providing evidence for interspecies communication. Here, we further confirmed that such communication occurs by showing that acyltransferase expression is induced by culture fluid obtained from diverse Gram-negative and -positive bacterial strains, including Escherichia coli, Salmonella Typhimurium, Bacillus subtilis, Staphylococcus aureus, Mycobacterium sp. strain JC1, and Mycobacterium smegmatis. A homologous acyltransferase encoded by PA5238 of P. aeruginosa was also induced by fluids obtained from P. aeruginosa as well as other bacterial strains, as observed for NCgl0350 of C. glutamicum. Because C. glutamicum is difficult to study using molecular approaches, the homologous gene PA5238 of P. aeruginosa was used to identify PA5309 as an upstream regulator of expression. A homologous D-amino acid dehydrogenase encoded by NCgl- 2909 of C. glutamicum was cloned based on amino acid similarity to PA5309, and its role in the regulation of NCgl0350 expression was confirmed. Moreover, NCgl2909 played positive roles in growth of C. glutamicum. Thus, we identified a D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in C. glutamicum.

Ribosome is responsible for protein synthesis and is able to monitor the sequence and structure of the nascent peptide. Such ability plays an important role in determining overall gene expression profile of the bacteria through ribosome stalling and rescuing. In this review, we briefly summarize our current understanding of the regulation of gene expression through ribosome stalling and rescuing in bacteria, as well as mechanisms that modulate ribosome activity. Understanding the mechanisms of how bacteria modulate ribosome activity will provide not only fundamental insights into bacterial gene regulation, but also new candidate targets for the development of novel antimicrobial agents.