Active extrusion is a key mechanism by which efflux pumps enable nosocomial pathogens to withstand exposure to antimicrobial compounds. In Acinetobacter baumannii, the AmvA efflux pump, belonging to the Major Facilitator Superfamily (MFS) and regulated by the TetR-family repressor AmvR, reduces susceptibility to disinfectants. Here, we investigated the broader functions of the AmvAR system beyond antimicrobial efflux. Our findings demonstrate that, in addition to conferring reduced susceptibility to disinfectants, the AmvAR system regulates the expression of cell surface components linked to pathogenic traits. Specifically, the A. baumannii AmvAR system contributes to motility and cell adhesion, thereby influencing biofilm formation. Loss of either the AmvA efflux pump or its transcriptional regulator AmvR significantly reduced biofilm development. Furthermore, disruption of regulatory control within this system led to a significant upregulation of the csuA pilus gene. Collectively, these results highlight the role of the AmvAR system in the pathogenesis of A. baumannii, underscoring its potential as a therapeutic target.

ABSTRACT Klebsiella pneumoniae is an opportunistic pathogen responsible for a wide range of healthcare-associated infections. In a recent work, we identified a novel Major Facilitator Superfamily efflux system in Acinetobacter baumannii , designated SxtPR, which mediates antimicrobial resistance and pathogenicity (M. Gaona, J. Corral, S. Campoy, J. Barbé, et al, Antimicrob Agents Chemother e0071224, 2024, https://doi.org/10.1128/aac.00712-24 ). Here, we report the identification and characterization of a homologous system in Klebsiella pneumoniae that plays a similar dual role in antimicrobial resistance and virulence.

Members of the S24 family of serine peptidases play central roles in bacterial and phage regulatory networks that respond to DNA damage. In most bacteria, the S24-family peptidase LexA functions as the master repressor of the SOS response, regulating the activity of genes involved in DNA repair and mutagenesis. LexA is known to be absent in some bacterial lineages, including genera of intracellular bacteria with substantial genomic reduction, but it has been shown that other S24-family peptidases can uptake its regulatory functions. In this work, we combine experimental and computational approaches to define the response to DNA damage in Francisella, a genus of facultative intracellular bacteria with moderate genome reduction that encodes a highly diverged LexA family protein. We use RNA sequencing to investigate the transcriptional response of Francisella hispaniensis to the DNA-damaging agent ciprofloxacin and reveal that canonical SOS genes are not induced upon DNA damage. Among the differentially expressed genes, we identify a gene encoding an S24-family peptidase that we term FddR (Francisella DNA-damage regulator). Electrophoretic mobility shift assays demonstrate that the product of this gene specifically binds to the palindromic sequence GTG-N11-CAC present in its promoter region, resulting in autoregulation. In silico analyses reveal that this S24-family peptidase is most likely a co-opted phage repressor that implements a DNA-damage response across the Francisella genus, regulating itself and divergently transcribed genes. Our results provide insights into the SOS repair system of the Francisella genus and its evolution, putting forward a mechanism for the gradual reconstruction of an SOS response network from self-contained divergently transcribed units.

Introduction:Acinetobacter baumannii is a highly adaptable opportunistic pathogen, notorious for its persistence in healthcare environments and its remarkable ability to withstand oxidative stress, antimicrobial treatments, and immune defenses. Methods:In this study, we characterized the role of the GntR family transcriptional regulator AUO97_RS16340 and its associated ABC efflux pump (AUO97_RS16355-RS16360-RS16365), previously identified as part of the OxyR regulon. Results:Through gain- and loss-of-function analyses, we demonstrate that this GntR regulator positively regulates the expression of the adjacent ABC efflux pump and plays a critical role in tolerance to H2O2-induced oxidative damage. Furthermore, we show that this efflux system contributes to multiple virulence-associated traits, including antimicrobial resistance, surface-associated motility, and virulence. Discussion:Our findings reveal a new efflux system that links oxidative stress adaptation, efflux-mediated antimicrobial susceptibility, and pathogenicity in A. baumannii strain ATCC 17978. Given the high conservation of the GntR transcriptional regulator and its associated efflux pump across A. baumannii genomes, these components represent promising therapeutic targets. Disrupting their activity could sensitize bacteria to oxidative stress and antimicrobials while attenuating virulence, providing a potential strategy to combat multidrug-resistant A. baumannii.

Acinetobacter baumannii and Enterobacter cloacae are phylogenetically distant Gram-negative bacterial pathogens that represent significant challenges in healthcare settings due to their remarkable ability to acquire antimicrobial resistance. This study investigates one of the most important efflux pump systems in A. baumannii, AdeABC-AdeRS, and identifies homologous components in E. cloacae. By constructing isogenic knockout mutants, we show that the AdeB pump component and the AdeR regulator are significant for antimicrobial resistance and pathogenicity in A. baumannii. Through in silico predictions, we identify homologs of AdeB and AdeR (ECL_01758 and ECL_01761, respectively) in E. cloacae. Notably, we demonstrate that while the inactivation of the E. cloacae gene encoding the AdeB protein does not impact on pathogenesis and only alters colistin susceptibility, a knockout mutant of the gene encoding the AdeR regulator significantly affects susceptibility to various antimicrobial classes, motility, and virulence. Additionally, we demonstrate that the AdeR regulators of A. baumannii and E. cloacae can functionally substitute for each other both in vitro and in vivo conditions. Electrophoretic mobility shift assays reveal that these regulators are capable of binding to the promoter regions of each other's species, where similar DNA motifs are present. Furthermore, cross-complementation tests show that the affected phenotypes in each species can be restored interchangeably. Moreover, phylogenomic analysis of previously published E.cloacae genomes and reconstructrion of ancestral states through the phylogenetic trees of the adeB and adeR genes suggest that these homologs are more likely derived from a common ancestor rather than through recent horizontal gene transfer. The findings of this work highlight that conserved regulatory functions concerning efflux pump expression can be maintained across species despite evolutionary divergence and open new perspectives for the control of bacterial infections.

Acinetobacter baumannii is a notorious opportunistic pathogen responsible for healthcare-associated infections worldwide. Efflux pumps play crucial roles in mediating antimicrobial resistance, motility, and virulence. In this study, we present the identification and characterization of the new A. baumannii efflux pump SxtP belonging to the MFS superfamily (major facilitator superfamily), along with its associated activator LysR-type transcriptional regulator (LTTR) SxtR, demonstrating their roles in sulfamethoxazole/trimethoprim (also known as co-trimoxazole or SXT) resistance, surface-associated motility and virulence.

Streptococcus suis serotype 2 is an important bacterial pathogen of swine, responsible for substantial economic losses to the swine industry worldwide. The knowledge on the pathogenesis of the infection caused by S. suis is still poorly known. It has been previously described that S. suis possesses at least one lipoprotein with double laminin and zinc (Zn)-binding properties, which was described in the literature as either laminin-binding protein (Lmb, as in the current study), lipoprotein 103, CDS 0330 or AdcAII. In the present study, the role of the Lmb in the pathogenesis of the infection caused by S. suis serotype 2 was dissected. Using isogenic mutants, results showed that Lmb does not play an important role in the laminin-binding activity of S. suis, even when clearly exposed at the bacterial surface. In addition, the presence of this lipoprotein does not influence bacterial adhesion to and invasion of porcine respiratory epithelial and brain endothelial cells and it does not increase the susceptibility of S. suis to phagocytosis. On the other hand, the Lmb was shown to play an important role as cytokine activator when tested in vitro with dendritic cells. Finally, this lipoprotein plays a critical role in Zn acquisition from the host environment allowing bacteria to grow in vivo. The significant lower virulence of the Lmb defective mutant may be related to a combination of a lower bacterial survival due to the incapacity to acquire Zn from their surrounding milieu and a reduced cytokine activation.

Acinetobacter baumannii is a pathogen of increasing clinical importance worldwide, especially given its ability to readily acquire resistance determinants. Motile strains of this bacterium can move by either or both of two types of motility: (i) twitching, driven by type IV pili, and (ii) surface-associated motility, an appendage-independent form of movement. A. baumannii strain MAR002 possesses both twitching and surface-associated motility. In this study, we isolated spontaneous rifampin-resistant mutants of strain MAR002 in which point mutations in the rpoB gene were identified that resulted in an altered motility pattern. Transcriptomic analysis of mutants lacking twitching, surface-associated motility, or both led to the identification of deregulated genes within each motility phenotype, based on their level of expression and their biological function. Investigations of the corresponding knockout mutants revealed several genes involved in the motility of A. baumannii strain MAR002, including two involved in twitching (encoding a minor pilin subunit and an RND [resistance nodulation division] component), one in surface-associated motility (encoding an amino acid permease), and eight in both (encoding RND and ABC components, the energy transducer TonB, the porin OprD, the T6SS component TagF, an IclR transcriptional regulator, a PQQ-dependent sugar dehydrogenase, and a putative pectate lyase). Virulence assays showed the reduced pathogenicity of mutants with impairments in both types of motility or in surface-associated motility alone. By contrast, the virulence of twitching-affected mutants was not affected. These results shed light on the key role of surface-associated motility and the limited role of twitching in the pathogenicity of A. baumannii.

Multidrug-Resistant (MDR) and Extensively Drug Resistant (XDR) Acinetobacter baumannii (Ab) represent a serious cause of healthcare-associated infections worldwide. Currently, the available treatment options are very restricted and colistin-based therapies are last-line treatments of these infections, even though colistin resistant (COLR) Ab have rarely been isolated yet. In bacteria, small non-coding RNAs (sRNAs) have been implicated in regulatory pathways of different biological functions, however, no knowledge exists about the sRNA role on the biological adaptation in COLRAb. Our study investigated two Italian XDR isogenic colistin-susceptible/resistant (COLS/R) Ab strain-pairs to discover new sRNA signatures. Comparative sRNA transcriptome (sRNAome) analyses were carried out by Illumina RNA-seq using both a Tru-Seq and a Short Insert library, whilst Ab ATCC 17978 and ACICU Reference Genome assembly, mapping, annotation and statistically significant differential expression (q-value ≤ 0.01) of the raw reads were performed by the Rockhopper tool. A computational filtering, sorting only similarly statistically significant differentially expressed (DE) sRNAs mapping on the same gene in both COLRAb isolates was conducted. COLR vs. COLS sRNAome, analyzed integrating the DE sRNAs obtained from the two different libraries, revealed some statistically significant DE sRNAs in COLRAb. In detail, we found: (i) two different under-expressed cis-acting sRNAs (AbsRNA1 and AbsRNA2) mapping in antisense orientation the 16S rRNA gene A1S_r01, (ii) one under-expressed cis-acting sRNA (AbsRNA3) targeting the A1S_2505 gene (hypothetical protein), (iii) one under-expressed microRNA-size small RNA fragment (AbsRNA4) and its pre-microAbsRNA4 targeting the A1S_0501 gene (hypothetical protein), (iv) as well as an over-expressed microRNA-size small RNA fragment (AbsRNA5) and its pre-microAbsRNA5 targeting the A1S_3097 gene (signal peptide). Custom TaqMan® probe-based real-time qPCRs validated the expression pattern of the selected sRNA candidates shown by RNA-seq. Furthermore, analysis on sRNA ΔA1S_r01, ΔA1S_2505 as well as the over-expressed A1S_3097 mutants revealed no effects on colistin resistance. Our study, for the first time, found the sRNAome signatures of clinical COLRAb with a computational prediction of their targets related to protein synthesis, host-microbe interaction and other different biological functions, including biofilm production, cell-cycle control, virulence, and antibiotic-resistance.

Twitching and swimming are two bacterial movements governed by pili and flagella. The present work identifies for the first time in the Gram-negative plant pathogen Ralstonia solanacearum a pilus-mediated chemotaxis pathway analogous to that governing flagellum-mediated chemotaxis. We show that regulatory genes in this pathway control all of the phenotypes related to pili, including twitching motility, natural transformation, and biofilm formation, and are also directly implicated in virulence, mainly during the first steps of the plant infection. Our results show that pili have a higher impact than flagella on the interaction of R. solanacearum with tomato plants and reveal new types of cross-talk between the swimming and twitching motility phenotypes: enhanced swimming in bacteria lacking pili and a role for the flagellum in root attachment.

Acinetobacter baumannii is a nosocomial pathogen that causes multi-drug resistant infections mainly in immunocompromised patients. Although this gram-negative species lacks flagella, it is able to move over wet surfaces through a not well characterized type of movement known as surface-associated motility. In this study we demonstrate through the inactivation of the A1S_2813 gene (coding a CheW-like protein) and recA (coding a DNA damage repair and recombination protein) that both genes are involved in the surface-associated motility and chemotaxis of A. baumannii ATCC 17978 strain. In addition, we also point out that the lack of either RecA or CheW-like proteins reduces its virulence in the Caenorhabditis elegans and the Galleria mellonella animal models. Furthermore, we show through co-immunoprecipitation assays that the CheW-like protein and RecA interact and that this interaction is abolished by the introduction of the mutation S97A in one of the domains of CheW-like protein that is structurally conserved in Salmonella enterica and necessary for the RecA-CheW interaction in this bacterial species. Finally, we show that the replacement of the wild-type CheW-like protein by that presenting the S97A mutation impairs surface-associated motility, chemotaxis and virulence of A. baumannii strain ATCC 17978.

Although the relationship between Acinetobacter baumannii efflux pumps and antimicrobial resistance is well documented, less is known about the involvement of these proteins in the pathogenicity of this nosocomial pathogen. In previous work, we identified the AbaQ major facilitator superfamily (MFS) efflux pump and demonstrated its participation in the motility and virulence of A. baumannii. In the present study, we examined the role in these processes of A. baumannii transporters belonging to different superfamilies of efflux pumps. Genes encoding known or putative permeases belonging to efflux pump superfamilies other than the MFS were selected, and the corresponding knockouts were constructed. The antimicrobial susceptibilities of these mutants were consistent with previously reported data. In mutants of A. baumannii strain ATCC 17978 carrying inactivated genes encoding the efflux pumps A1S_2736 (resistance nodulation division [RND]), A1S_3371 (multidrug and toxic compound extrusion [MATE]), and A1S_0710 (small multidrug resistance [SMR]), as well as the newly described ATP-binding cassette (ABC) permeases A1S_1242 and A1S_2622, both surface-associated motility and virulence were reduced compared to the parental strain. However, inactivation of the genes encoding the known ABC permeases A1S_0536 and A1S_1535, the newly identified putative ABC permeases A1S_0027 and A1S_1057, or the proteobacterial antimicrobial compound efflux (PACE) transporters A1S_1503 and A1S_2063 had no effects on bacterial motility or virulence. Our results demonstrate the involvement of antimicrobial transporters belonging at least to five of the six known efflux pump superfamilies in both surface- associated motility and virulence in A. baumannii ATCC 17978.

14 Acinetobacter baumannii has emerged as an important multidrug-resistant nosocomial 15 pathogen. In previous work, we identified a putative MFS transporter, 16 AU097_RS17040, involved in the pathogenicity of A. baumannii (Pérez-Varela et al., 17 Infect Immun. 2017). In this study, we analyzed the susceptibility to diverse 18 antimicrobial agents of A. baumannii cells defective in this transporter, referred to 19 herein as AbaQ. Our results showed that AbaQ is mainly involved in the extrusion of 20 quinolone-type drugs in A. baumannii. 21 22 AAC Accepted Manuscript Posted Online 25 June 2018 Antimicrob. Agents Chemother. doi:10.1128/AAC.00906-18 Copyright © 2018 American Society for Microbiology. All Rights Reserved. on Jne 7, 2018 by M O O D Y M E D IC A L IB R A R Y httpaac.asm .rg/ D ow nladed fom

Acinetobacter baumannii has emerged as an important multidrug-resistant nosocomial pathogen. In previous work, we identified a putative MFS transporter, AU097_RS17040, involved in the pathogenicity of A. baumannii (M.

Colistin is a crucial last-line drug used for the treatment of life-threatening infections caused by multidrug-resistant strains of the Gram-negative bacterium Acinetobacter baumannii However, colistin-resistant A. baumannii isolates can still be isolated following failed colistin therapy. Resistance is most often mediated by the addition of phosphoethanolamine (pEtN) to lipid A by PmrC, following missense mutations in the pmrCAB operon encoding PmrC and the two-component signal transduction system PmrA/PmrB. We recovered a pair of A. baumannii isolates from a single patient before (6009-1) and after (6009-2) failed colistin treatment. These strains displayed low and very high levels of colistin resistance (MICs, 8 to 16 μg/ml and 128 μg/ml), respectively. To understand how increased colistin resistance arose, we sequenced the genome of each isolate, which revealed that 6009-2 had an extra copy of the insertion sequence element ISAba125 within a gene encoding an H-NS family transcriptional regulator. To confirm the role of H-NS in colistin resistance, we generated an hns deletion mutant in 6009-1 and showed that colistin resistance increased upon the deletion of hns We also provided 6009-2 with an intact copy of hns and showed that the strain was no longer resistant to high concentrations of colistin. Transcriptomic analysis of the clinical isolates identified more than 150 genes as being differentially expressed in the colistin-resistant hns mutant 6009-2. Importantly, the expression of eptA, encoding a second lipid A-specific pEtN transferase but not pmrC, was increased in the hns mutant. This is the first time an H-NS family transcriptional regulator has been associated with a pEtN transferase and colistin resistance.

Acinetobacter baumannii is a major cause of antibiotic-resistant nosocomial infections worldwide. In this study, several rifampin-resistant spontaneous mutants obtained from the A. baumannii ATCC 17978 strain that differed in their point mutations in the rpoB gene, encoding the β-subunit of the RNA polymerase, were isolated. All the mutants harboring amino acid substitutions in position 522 or 540 of the RpoB protein were impaired in surface-associated motility and had attenuated virulence in the fertility model of Caenorhabditis elegans The transcriptional profile of these mutants included six downregulated genes encoding proteins homologous to transporters and metabolic enzymes widespread among A. baumannii clinical isolates. The construction of knockout mutants in each of the six downregulated genes revealed a significant reduction in the surface-associated motility and virulence of four of them in the A. baumannii ATCC 17978 strain, as well as in the virulent clinical isolate MAR002. Taken together, our results provide strong evidence of the connection between motility and virulence in this multiresistant nosocomial pathogen.

In addition to its role in DNA damage repair and recombination, the RecA protein, through its interaction with CheW, is involved in swarming motility, a form of flagella-dependent movement across surfaces. In order to better understand how SOS response modulates swarming, in this work the location of RecA and CheW proteins within the swarming cells has been studied by using super-resolution microscopy. Further, and after in silico docking studies, the specific RecA and CheW regions associated with the RecA-CheW interaction have also been confirmed by site-directed mutagenesis and immunoprecipitation techniques. Our results point out that the CheW distribution changes, from the cell poles to foci distributed in a helical pattern along the cell axis when SOS response is activated or RecA protein is overexpressed. In this situation, the CheW presents the same subcellular location as that of RecA, pointing out that the previously described RecA storage structures may be modulators of swarming motility. Data reported herein not only confirmed that the RecA-CheW pair is essential for swarming motility but it is directly involved in the CheW distribution change associated to SOS response activation. A model explaining not only the mechanism by which DNA damage modulates swarming but also how both the lack and the excess of RecA protein impair this motility is proposed.

ABSTRACT Acinetobacter baumannii , a worldwide emerging nosocomial pathogen, acquires antimicrobial resistances in response to DNA-damaging agents, which increase the expression of multiple error-prone DNA polymerase components. Here we show that the aminocoumarin novobiocin, which inhibits the DNA damage response in Gram-positive bacteria, also inhibits the expression of error-prone DNA polymerases in this Gram-negative multidrug-resistant pathogen and, consequently, its potential acquisition of antimicrobial resistance through DNA damage-induced mutagenesis.

The effect of antimicrobials on SOS-mediated mutagenesis induction depends on the bacterial species and the antimicrobial group. In this work, we studied the effect of different families of antimicrobial agents used in clinical therapy against Acinetobacter baumannii in the induction of mutagenesis in this multiresistant Gram-negative pathogen. The data showed that ciprofloxacin and tetracycline induce SOS-mediated mutagenesis, whereas colistin and meropenem, which are extensively used in clinical therapy, do not.

OBJECTIVESColistin remains a last-line treatment for MDR Acinetobacter baumannii and combined use of colistin and carbapenems has shown synergistic effects against MDR strains. In order to understand the bacterial responses to these antibiotics, we analysed the transcriptome of A. baumannii following exposure to each.METHODSRNA sequencing was employed to determine changes in the transcriptome following treatment with colistin and doripenem, both alone and in combination, using an in vitro pharmacokinetics (PK)/pharmacodynamics model to mimic the PK of both antibiotics in patients.RESULTSAfter treatment with colistin (continuous infusion at 2 mg/L), >400 differentially regulated genes were identified, including many associated with outer membrane biogenesis, fatty acid metabolism and phospholipid trafficking. No genes were differentially expressed following treatment with doripenem (Cmax 25 mg/L, t1/2 1.5 h) for 15 min, but 45 genes were identified as differentially expressed after 1 h of growth under this condition. Treatment of A. baumannii with both colistin and doripenem together for 1 h resulted in >450 genes being identified as differentially expressed. More than 70% of these gene expression changes were also observed following colistin treatment alone.CONCLUSIONSThese data suggest that colistin causes gross damage to the outer membrane, facilitates lipid exchange between the inner and outer membrane and alters the normal asymmetric outer membrane composition. The transcriptional response to colistin was highly similar to that observed for an LPS-deficient strain, indicating that many of the observed changes are responses to outer membrane instability resulting from LPS loss.