RecA levels modulate biofilm development in Acinetobacter baumannii

AbstractInfections caused by Acinetobacter baumannii, a Gram-negative opportunistic pathogen, are difficult to eradicate due to the bacterium’s propensity to quickly gain antibiotic resistances and form protective bacterial multicellular communities known as biofilms. The A. baumannii DNA damage response (DDR) mediates antibiotic resistance acquisition and regulates RecA in an atypical fashion; both RecALow and RecAHigh cell types are formed in response to DNA damage. In this study, we show that RecA levels modulate biofilm development, formation and dispersal, through bfmR, the global biofilm regulator. RecA loss results in surface attachment and prominent biofilms while elevated RecA leads to diminished attachment and dispersal. Recalcitrance to treatment may be explained by DDR induction, common during infection, and the balance between biofilm maintenance in low RecA cells, and increased mutagenesis and dispersal to reach new niches in high RecA cells. These data highlight the importance of understanding fundamental biology to better treat bacterial infections.ImpactThe mechanism of biofilm formation and dispersal in A. baumannii, shown here to depend on RecA levels, contributes to the understanding of recalcitrant infections caused by this important pathogen.

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Lon Protease Has Multifaceted Biological Functions inAcinetobacter baumannii

Journal of Bacteriology ◽

10.1128/jb.00536-18 ◽

2018 ◽

Vol 201 (2) ◽

Cited By ~ 5

Author(s):

Carly Ching ◽

Brendan Yang ◽

Chineme Onwubueke ◽

David Lazinski ◽

Andrew Camilli ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Developmental Process ◽

Cell Envelope ◽

Opportunistic Pathogen ◽

Lon Protease ◽

Content Type ◽

Hospital Acquired Infections ◽

Biofilm Development ◽

Hospital Acquired

ABSTRACTAcinetobacter baumanniiis a Gram-negative opportunistic pathogen that is known to survive harsh environmental conditions and is a leading cause of hospital-acquired infections. Specifically, multicellular communities (known as biofilms) ofA. baumanniican withstand desiccation and survive on hospital surfaces and equipment. Biofilms are bacteria embedded in a self-produced extracellular matrix composed of proteins, sugars, and/or DNA. Bacteria in a biofilm are protected from environmental stresses, including antibiotics, which provides the bacteria with selective advantage for survival. Although some gene products are known to play roles in this developmental process inA. baumannii, mechanisms and signaling remain mostly unknown. Here, we find that Lon protease inA. baumanniiaffects biofilm development and has other important physiological roles, including motility and the cell envelope. Lon proteases are found in all domains of life, participating in regulatory processes and maintaining cellular homeostasis. These data reveal the importance of Lon protease in influencing keyA. baumanniiprocesses to survive stress and to maintain viability.IMPORTANCEAcinetobacter baumanniiis an opportunistic pathogen and is a leading cause of hospital-acquired infections.A. baumanniiis difficult to eradicate and to manage, because this bacterium is known to robustly survive desiccation and to quickly gain antibiotic resistance. We sought to investigate biofilm formation inA. baumannii, since much remains unknown about biofilm formation in this bacterium. Biofilms, which are multicellular communities of bacteria, are surface attached and difficult to eliminate from hospital equipment and implanted devices. Our research identifies multifaceted physiological roles for the conserved bacterial protease Lon inA. baumannii. These roles include biofilm formation, motility, and viability. This work broadly affects and expands understanding of the biology ofA. baumannii, which will permit us to find effective ways to eliminate the bacterium.

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Comparative biofilm assays using Enterococcus faecalis OG1RF identify new determinants of biofilm formation

10.1101/2021.02.24.432758 ◽

2021 ◽

Author(s):

Julia L. E. Willett ◽

Jennifer L. Dale ◽

Lucy M. Kwiatkowski ◽

Jennifer L. Powers ◽

Michelle L. Korir ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Growth Medium ◽

Biofilm Formation ◽

Time Course ◽

Opportunistic Pathogen ◽

Genetic Determinants ◽

Experimental Conditions ◽

Biofilm Reactors ◽

Biofilm Architecture ◽

Biofilm Development

AbstractEnterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants. We quantified biofilm cells and used fluorescence microscopy to visualize biofilms formed by 6 Tn mutants identified using TnSeq and found that disrupting these genes (OG1RF_10350, prsA, tig, OG1RF_10576, OG1RF_11288, and OG1RF_11456) leads to significant time- and medium-dependent changes in biofilm architecture. Structural predictions revealed potential roles in cell wall homeostasis for OG1RF_10350 and OG1RF_11288 and signaling for OG1RF_11456. Additionally, we identified growth medium-specific hallmarks of OG1RF biofilm morphology. This study demonstrates how E. faecalis biofilm architecture is modulated by growth medium and experimental conditions, and identifies multiple new genetic determinants of biofilm formation.ImportanceE. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients. We employed a comprehensive approach to analysis of biofilm determinants by combining TnSeq primary screens with secondary phenotypic validation using diverse biofilm assays. This enabled identification of numerous core (important under many conditions) and accessory (important under specific conditions) biofilm determinants in E. faecalis OG1RF. We found multiple genes whose disruption results in drastic changes to OG1RF biofilm morphology. These results expand our understanding of the genetic requirements for biofilm formation in E. faecalis that affect the time course of biofilm development as well as the response to specific nutritional conditions.

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Correlation of biofilm formation and antibiotic resistance among clinical and soil isolates of Acinetobacter baumannii in Iraq

Acta Microbiologica et Immunologica Hungarica ◽

10.1556/030.66.2019.026 ◽

2019 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Pakhshan A. Hassan ◽

Adel K. Khider

Keyword(s):

Acinetobacter Baumannii ◽

Congo Red ◽

Biofilm Formation ◽

Opportunistic Pathogen ◽

Microtiter Plate ◽

Test Tube ◽

Clinical Isolates ◽

Diffusion Method ◽

Biochemical Tests ◽

Disk Diffusion Method

Acinetobacter baumannii is an opportunistic pathogen that is reported as a major cause of nosocomial infections. The aim of this study was to investigate the biofilm formation by A. baumannii clinical and soil isolates, to display their susceptibility to 11 antibiotics and to study a possible relationship between formation of biofilm and multidrug resistance. During 8 months period, from June 2016 to January 2017, a total of 52 clinical and 22 soil isolates of A. baumannii were collected and identified through conventional phenotypic, chromo agar, biochemical tests, API 20E system, and confirmed genotypically by PCR for blaOXA-51-like gene. Antibiotic susceptibility of isolates was determined by standard disk diffusion method according to Clinical and Laboratory Standard Institute. The biofilm formation was studied using Congo red agar, test tube, and microtiter plate methods. The clinical isolates were 100% resistance to ciprofloxacin, ceftazidime, piperacillin, 96.15% to gentamicin, 96.15% to imipenem, 92.31% to meropenem, and 78.85% to amikacin. The soil A. baumannii isolates were 100% sensitive to imipenem, meropenem, and gentamicin, and 90.1% to ciprofloxacin. All A. baumannii isolates (clinical and soil) were susceptible to polymyxin B. The percentage of biofilm formation in Congo red agar, test tube, and microtiter plate assays was 10.81%, 63.51%, and 86.48%, respectively. More robust biofilm former population was mainly among non-MDR isolates. Isolates with a higher level of resistance tended to form weaker biofilms. The soil isolates exhibited less resistance to antibiotics than clinical isolates. However, the soil isolates produce stronger biofilms than clinical isolates.

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Biofilm Formation and Detection of Fluoroquinolone- and Carbapenem-Resistant Genes in Multidrug-Resistant Acinetobacter baumannii

Canadian Journal of Infectious Diseases and Medical Microbiology ◽

10.1155/2019/3454907 ◽

2019 ◽

Vol 2019 ◽

pp. 1-5 ◽

Cited By ~ 7

Author(s):

María-Guadalupe Avila-Novoa ◽

Oscar-Alberto Solís-Velázquez ◽

Daniel-Eduardo Rangel-López ◽

Jean-Pierre González-Gómez ◽

Pedro-Javier Guerrero-Medina ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Opportunistic Pathogen ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Low Grade ◽

Carbapenem Resistant ◽

Hospital Environments ◽

Potential Association ◽

Clinical Environments

Acinetobacter baumannii is an important opportunistic pathogen that shows resistance to cephalosporins, penicillins, carbapenems, fluoroquinolones, and aminoglycosides, the multiresistance being associated with its ability to form biofilms in clinical environments. The aim of this study was to determine biofilm formation and its potential association with genes involved in antibiotic resistance mechanisms of A. baumannii isolates of different clinical specimens. We demonstrated 100% of the A. baumannii isolates examined to be multidrug resistant (MDR), presenting a 73.3% susceptibility to cefepime and a 53.3% susceptibility to ciprofloxacin. All A. baumannii isolates were positive for blaOXA-51, 33.3% being positive for blaOXA-23 and ISAba1, and 73.3% being positive for gyrA. We found 86.6% of A. baumannii strains to be low-grade biofilm formers and 13.3% to be biofilm negative; culturing on Congo red agar (CRA) plates revealed that 73.3% of the A. baumannii isolates to be biofilm producers, while 26.6% were not. These properties, combined with the role of A. baumannii as a nosocomial pathogen, increase the probability of A. baumannii causing nosocomial infections and outbreaks as a complication during therapeutic treatments and emphasize the need to control A. baumannii biofilms in hospital environments.

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Phage φAB6-Borne Depolymerase Combats Acinetobacter baumannii Biofilm Formation and Infection

Antibiotics ◽

10.3390/antibiotics10030279 ◽

2021 ◽

Vol 10 (3) ◽

pp. 279

Author(s):

Md. Shahed-Al-Mahmud ◽

Rakesh Roy ◽

Febri Gunawan Sugiokto ◽

Md. Nazmul Islam ◽

Ming-Der Lin ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Bacterial Infections ◽

Capsular Polysaccharide ◽

Foley Catheter ◽

Inhibition Zone ◽

Control Group ◽

Bacterial Cells ◽

Bacterial Lawn

Biofilm formation is one of the main causes of increased antibiotic resistance in Acinetobacter baumannii infections. Bacteriophages and their derivatives, such as tail proteins with depolymerase activity, have shown considerable potential as antibacterial or antivirulence agents against bacterial infections. Here, we gained insights into the activity of a capsular polysaccharide (CPS) depolymerase, derived from the tailspike protein (TSP) of φAB6 phage, to degrade A. baumannii biofilm in vitro. Recombinant TSP showed enzymatic activity and was able to significantly inhibit biofilm formation and degrade formed biofilms; as low as 0.78 ng, the inhibition zone can still be formed on the bacterial lawn. Additionally, TSP inhibited the colonization of A. baumannii on the surface of Foley catheter sections, indicating that it can be used to prevent the adhesion of A. baumannii to medical device surfaces. Transmission and scanning electron microscopy demonstrated membrane leakage of bacterial cells treated with TSP, resulting in cell death. The therapeutic effect of TSP in zebrafish was also evaluated and the results showed that the survival rate was significantly improved (80%) compared with that of the untreated control group (10%). Altogether, we show that TSP derived from φAB6 is expected to become a new antibiotic against multi-drug resistant A. baumannii and a biocontrol agent that prevents the formation of biofilms on medical devices.

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A SIR2 family protein impacts biofilm formation by post-translational modifications in Acinetobacter baumannii

10.5194/biofilms9-127 ◽

2020 ◽

Author(s):

Brandon Robin ◽

Sebastien Massier ◽

Anaïs Potron ◽

Jean-Baptiste Vuillemenot ◽

Salomé Sauvage ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Therapeutic Targets ◽

World Health ◽

Protein Acetylation ◽

Acinetobacter Baumanii ◽

Post Translational Modifications ◽

Bacterial Physiology ◽

Global Dynamic ◽

Biofilm Development

Acinetobacter baumannii is one of the most problematic opportunist pathogen responsible for many infections worldwide (1). Besides its high capacities to acquire antibiotic resistance mechanisms, it also presents high adhesion abilities on any types of abiotic or living surfaces leading to biofilm development, a mode of growth conferring an additional protection against various treatments and allowing the infection relapse (2). A. baumannii has been recently ranked on the global priority pathogens list established by the World Health Organization for which there is an urgent need for new treatments. One interesting way to identify new therapeutic targets to eradicate this pathogen is the characterization of its post-translational modifications (PTMs) (3). The functions and extents of PTMs remain largely unknown in prokaryotic cells compared to eukaryotic cells. Lysine acetylation is an attractive and prevalent PTM in bacteria. An increasing number of investigations have been dedicated to identify acetylated proteins by proteomics. Some studies have shown that acetylation can play a pivotal role in bacterial virulence, resistance, or biofilm (4). Enzymes involved in acetylation addition (lysine acetyltranferase KAT) or removal (lysine deacetylase KDAC) would provide a better mechanistic understanding of bacterial physiology and therefore could be considered as potential therapeutic targets. So far, little information is available on these enzymes in A. baumannii (5). Recently, in a global dynamic proteome study of A. baumannii ATCC 17978 strain grown in sessile mode, we highlighted the highest protein fold change for a protein belonging to the Sir2-like family which may possess a KDAC activity (6). The aim of the current study was to evaluate the involvement of this protein in A. baumannii physiology. For this purpose, a gene deletion approach was carried out to perform different phenotype tests (drugs and oxidative stress resistance, virulence assays, motility and biofilm formation) on wild-type and mutant strains. We compared, in biofilm mode of growth, acetylomes of the WT and the mutant. Our results demonstrated more than twice acetylated proteins in mutant in comparison to the WT. Of interest, biofilm formation in mutant was sensibly decreased. These different results suggest a potential involvement of this protein in A. baumannii biofilm formation. &#160; (1) Antunes et al. Acinetobacter baumannii: evolution of a global pathogen. Pathog. Dis. 71(2014), 292-301. (2) Espinal et al. Effect of biofilm formation on the survival of Acinetobacter baumannii on dry surfaces. J. Hosp. Infect. 80(2012), 56&#8211;60. (3) Richters. Targeting protein arginine methyltransferase 5 in disease. Future Med. Chem. 9(2017), 2081-2098. (4) VanDrisse and Escalante-Semerena. Protein acetylation in bacteria. Annu. Rev. Microbiol. 73(2019), 111-132. (5) Carabetta and Cristea. Regulation, function, and detection of protein acetylation in bacteria. J. Bacteriol. 199(2017), e00107-17. (6) Kentache et al. Global dynamic proteome study of a pellicle-forming Acinetobacter baumanii strain. Mol. Cell. Proteomics. 16(2017), 100-112.

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Differential effect of hypoxia on etoposide-induced DNA damage response and p53 regulation in different cell types

Journal of Cellular Physiology ◽

10.1002/jcp.24409 ◽

2013 ◽

Vol 228 (12) ◽

pp. 2365-2376 ◽

Cited By ~ 14

Author(s):

Audrey Sermeus ◽

Magali Rebucci ◽

Maude Fransolet ◽

Lionel Flamant ◽

Déborah Desmet ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Differential Effect ◽

Cell Types ◽

Damage Response ◽

Different Cell Types

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AbaM Regulates Quorum Sensing, Biofilm Formation and Virulence in Acinetobacter baumannii

Journal of Bacteriology ◽

10.1128/jb.00635-20 ◽

2021 ◽

Author(s):

Mario López-Martín ◽

Jean-Frédéric Dubern ◽

Morgan R. Alexander ◽

Paul Williams

Keyword(s):

Quorum Sensing ◽

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Plant Pathogens ◽

Galleria Mellonella ◽

Homoserine Lactone ◽

Infection Model ◽

Wild Type ◽

Surface Motility ◽

Biofilm Development

Acinetobacter baumannii possesses a single divergent luxR/luxI-type quorum sensing (QS) locus named abaR/abaI. This locus also contains a third gene located between abaR and abaI which we term abaM that codes for an uncharacterized member of the RsaM protein family known to regulate N-acylhomoserine lactone (AHL) dependent QS in other β- and γ-proteobacteria. Here we show that disruption of abaM via a T26 insertion in A. baumannii strain AB5075 resulted in increased production of N-(3-hydroxydodecanoyl)-L-homoserine lactone (OHC12) and enhanced surface motility and biofilm formation. In contrast to the wild type and abaI::T26 mutant, the virulence of the abaM::T26 mutant was completely attenuated in a Galleria mellonella infection model. Transcriptomic analysis of the abaM::T26 mutant revealed that AbaM differentially regulates at least 76 genes including the csu pilus operon and the acinetin 505 lipopeptide biosynthetic operon, that are involved in surface adherence, biofilm formation and virulence. A comparison of the wild type, abaM::T26 and abaI::T26 transcriptomes, indicates that AbaM regulates ∼21% of the QS regulon including the csu operon. Moreover, the QS genes (abaI/abaR) were among the most upregulated in the abaM::T26 mutant. A. baumannii lux-based abaM reporter gene fusions revealed that abaM expression is positively regulated by QS but negatively auto-regulated. Overall, the data presented in this work demonstrates that AbaM plays a central role in regulating A. baumannii QS, virulence, surface motility and biofilm formation. Importance Acinetobacter baumanni is a multi-antibiotic resistant pathogen of global healthcare importance. Understanding Acinetobacter virulence gene regulation could aid the development of novel anti-infective strategies. In A. baumannii, the abaR and abaI genes that code for the receptor and synthase components of an N-acylhomoserine (AHL) lactone-dependent quorum sensing system (QS) are separated by abaM. Here we show that although mutation of abaM increased AHL production, surface motility and biofilm development, it resulted in the attenuation of virulence. AbaM was found to control both QS-dependent and QS-independent genes. The significance of this work lies in the identification of AbaM, an RsaM ortholog known to control virulence in plant pathogens, as a modulator of virulence in a human pathogen.

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Function of BriC Peptide in the Pneumococcal Competence and Virulence Portfolio

10.1101/245902 ◽

2018 ◽

Cited By ~ 1

Author(s):

Surya D. Aggarwal ◽

Rory Eutsey ◽

Jacob West-Roberts ◽

Arnau Domenech ◽

Wenjie Xu ◽

...

Keyword(s):

Murine Model ◽

Biofilm Formation ◽

Opportunistic Pathogen ◽

Nasopharyngeal Colonization ◽

Point Of Entry ◽

Abiotic Surfaces ◽

Biofilm Development

AbstractStreptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that causes otitis media, sinusitis, pneumonia, meningitis and sepsis. The progression to this pathogenic lifestyle is preceded by asymptomatic colonization of the nasopharynx. This colonization is associated with biofilm formation; the competence pathway influences the structure and stability of biofilms. However, the molecules that link the competence pathway to biofilm formation are unknown. Here, we describe a new competence-induced gene, called briC, and demonstrate that its product promotes biofilm development and stimulates colonization in a murine model. We show that expression of briC is induced by the master regulator of competence, ComE. Whereas briC does not substantially influence early biofilm development on abiotic surfaces, it significantly impacts later stages of biofilm development. Specifically, briC expression leads to increases in biofilm biomass and thickness at 72h. Consistent with the role of biofilms in colonization, briC promotes nasopharyngeal colonization in the murine model. The function of BriC appears to be conserved across pneumococci, as comparative genomics reveal that briC is widespread across isolates. Surprisingly, many isolates, including strains from clinically important PMEN1 and PMEN14 lineages, which are widely associated with colonization, encode a long briC promoter. This long form captures an instance of genomic plasticity and functions as a competence-independent expression enhancer that may serve as a precocious point of entry into this otherwise competence-regulated pathway. Moreover, overexpression of briC by the long promoter fully rescues the comE-deletion induced biofilm defect in vitro, and partially in vivo. These findings indicate that BriC may bypass the influence of competence in biofilm development and that such a pathway may be active in a subset of pneumococcal lineages. In conclusion, BriC is a part of the complex molecular network that connects signaling of the competence pathway to biofilm development and colonization.

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The SiaABC threonine phosphorylation pathway controls biofilm formation in response to carbon availability in Pseudomonas aeruginosa

PLoS ONE ◽

10.1371/journal.pone.0241019 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0241019

Author(s):

Wee-Han Poh ◽

Jianqing Lin ◽

Brendan Colley ◽

Nicolai Müller ◽

Boon Chong Goh ◽

...

Keyword(s):

Biofilm Formation ◽

Critical Role ◽

Dynamic Modelling ◽

Opportunistic Pathogen ◽

Crystallographic Analysis ◽

Carbon Substrate ◽

Carbon Availability ◽

Cellular Aggregation ◽

Biofilm Development

The critical role of bacterial biofilms in chronic human infections calls for novel anti-biofilm strategies targeting the regulation of biofilm development. However, the regulation of biofilm development is very complex and can include multiple, highly interconnected signal transduction/response pathways, which are incompletely understood. We demonstrated previously that in the opportunistic, human pathogen P. aeruginosa, the PP2C-like protein phosphatase SiaA and the di-guanylate cyclase SiaD control the formation of macroscopic cellular aggregates, a type of suspended biofilms, in response to surfactant stress. In this study, we demonstrate that the SiaABC proteins represent a signal response pathway that functions through a partner switch mechanism to control biofilm formation. We also demonstrate that SiaABCD functionality is dependent on carbon substrate availability for a variety of substrates, and that upon carbon starvation, SiaB mutants show impaired dispersal, in particular with the primary fermentation product ethanol. This suggests that carbon availability is at least one of the key environmental cues integrated by the SiaABCD system. Further, our biochemical, physiological and crystallographic data reveals that the phosphatase SiaA and its kinase counterpart SiaB balance the phosphorylation status of their target protein SiaC at threonine 68 (T68). Crystallographic analysis of the SiaA-PP2C domain shows that SiaA is present as a dimer. Dynamic modelling of SiaA with SiaC suggested that SiaA interacts strongly with phosphorylated SiaC and dissociates rapidly upon dephosphorylation of SiaC. Further, we show that the known phosphatase inhibitor fumonisin inhibits SiaA mediated phosphatase activity in vitro. In conclusion, the present work improves our understanding of how P. aeuruginosa integrates specific environmental conditions, such as carbon availability and surfactant stress, to regulate cellular aggregation and biofilm formation. With the biochemical and structural characterization of SiaA, initial data on the catalytic inhibition of SiaA, and the interaction between SiaA and SiaC, our study identifies promising targets for the development of biofilm-interference drugs to combat infections of this aggressive opportunistic pathogen.

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