Histone-like Nucleoid-structuring Protein (H-NS) Regulatory Role in Antibiotic Resistance in Acinetobacter Baumannii

doi:10.21203/rs.3.rs-690008/v1

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Histone-like Nucleoid-structuring Protein (H-NS) Regulatory Role in Antibiotic Resistance in Acinetobacter Baumannii

https://doi.org/10.21203/rs.3.rs-690008/v1

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In the multidrug resistant (MDR) pathogen Acinetobacter baumannii the global repressor H-NS was shown to modulate the expression of genes related to quorum sensing, biosynthetic pathways, cell adhesion, virulence, and autotransporters. In addition, H-NS is involved in stress response and its inactivation results in an increased resistance to colistin, and in a hypermotile phenotype an altered stress response. To further contribute to the knowledge of this key transcriptional regulator in A. baumannii behavior, we studied the role of H-NS in antimicrobial resistance.

Using two well characterized A. baumannii model strains with distinctive resistance profile and pathogenicity traits (AB5075 and A118), complementary transcriptomic and phenotypic approaches were used to study the role of H-NS in antimicrobial resistance, biofilm and quorum sensing gene expression.

An increased expression of genes associated with β-lactam resistance, aminoglycosides, quinolones, chloramphenicol, trimethoprim and sulfonamides resistance in the Δhns mutant background was observed. Genes codifying for efflux pumps were also up-regulated, with the exception of adeFGH. The wild-type transcriptional level was restored in the complemented strain. In addition, the expression of biofilm related genes and biofilm production was lowered when the transcriptional repressor was absent. The quorum network genes aidA, abaI, kar and fadD were up-regulated in Δhns mutant strains.

Overall, our results showed the complexity and scope of the regulatory network control by H-NS (genes involved in antibiotic resistance and persistence). These observations brings us one step closer to understanding the regulatory role of hns to combat A. baumannii infections.

Scientific Communication

Biotechnology and Bioengineering

Acinetobacter baumannii

H-NS

stress

antibiotic resistance

efflux pumps

biofilm

quorum sensing

Transcription, as a complex obligated process for gene expression, provides multiple targets for antimicrobial discovery. Among them, obvious candidates are transcriptional factors and regulators. While RNA polymerase and transcriptional factors are highly conserved across the bacterial kingdom, regulators can be more specific to each bacterium that must adapt to unique environments. Acinetobacter baumannii readily adjusts to the hostile environments it finds when it colonizes and infects different human body niches. A critical aspect of the adaptation strategies to survive and thrive in harsh conditions is the fine tuning of gene expression through the timely action of transcriptional regulators. Researchers have explored the regulation of antibiotic resistance and identified several two-component systems (TCSs) responsible for resistance to multiple antimicrobials through different mechanisms like regulation of RND-type efflux pumps ^{1, 2 3, 4}.

A transcription factor that has attracted attention is H-NS, which controls expression of many functions in bacteria, including adherence to biotic surfaces, quorum sensing, biofilm formation, and metabolism pathways ^{5,6 7–9}. In A. baumannii, H-NS regulates the expression of genes involved in a variety of biological processes like transport (e.g., autotransporters, type-VI secretion systems) and metabolism (e.g., phenylacetic acid degradation pathway) ⁷. Interestingly, H-NS inactivation, reduces levels of resistance to colistin ⁸, an antibiotic reintroduced to treat multidrug resistant (MDR) A. baumannii infections. H-NS is also responsible for protecting against stress; it alleviates the deleterious effects produced by metallo-β-lactamase toxic precursors and protects against DNA-damaging agents such as mitomycin C and levofloxacin ¹⁰. A recently described function of H-NS is the repression of the exopolysaccharide poly-N-acetylglucosamine synthesis and the expression of a protein belonging to the CsgG/HfaB family ¹¹. The expression of the A. baumannii H-NS is down regulated in environments containing HSA or HSA-containing fluids ^{12, 13}. In sum, its own regulatable nature strongly suggests that H-NS plays a fundamental role in A. baumannii’s responses and adaptation to the conditions within the human body.

In this article, we demonstrate H-NS's role in regulating expression of genes related to antibiotic resistance, biofilm formation, and quorum sensing in the multidrug-resistant, hypervirulent A. baumannii AB5075 and the susceptible, moderately virulent A. baumannii A118 strains.

Transcriptome analysis of A. baumannii strain AB5075 and AB5075 Δhns revealed a differential expression profile of 183 genes with an FDR-adjusted P-value < 0.05 and log2-fold change >1. Among the differentially expressed-genes (DEGs), we identified genes associated with motility, biofilm formation, quorum sensing, antibiotic resistance, metabolism, and stress response (Supplementary Table S1). As the aim of the present work is to identify the possible role of H-NS in antibiotic resistance, only those genes, directly or indirectly, associated with antibiotic resistance were examined (Supplementary Table S2).

H-NS plays a role in modulating the expression of antibiotic resistance associated genes

RNA-seq data analyzed showed that the expression of genes related to resistance to β-lactams, aminoglycosides, quinolones, chloramphenicol, trimethoprim, sulfonamides, and colistin were up-regulated in AB5075 Δhns strain (Figure 1A). On the other hand, genes related to tetracycline resistance were down-regulated in the Δhns strain compare to the parental strain. To confirm the RNA-seq results, transcript analysis of A. baumannii AB5075, AB5075 Δhns and AB5075 Δhns pMBLe-hns cells was performed, showing an enhanced expression on seven out of eight genes associated with β-lactam resistance in AB5075 Δhns mutant strain (Figure 1B and Supplementary Figure S1A). In addition, the increased expression of all the selected genes associated with aminoglycoside, quinolone, chloramphenicol, trimethoprim and sulfonamide resistance in AB5075 Δhns strain was confirmed by quantitative RT-PCR (qRT-PCR) assays (Figure 1B and Supplementary Figure S1A). Consistently, the mutant strain containing the plasmid pMBLe-hns, which expresses a wild-type copy of hns under the control of own promoter, rescued the expression for all resistance-related genes tested to wild type levels (Figure 1 B). These results provide evidence indicating that H-NS is involved in modulating the expression of resistance-related genes.

In order to further expand our knowledge about antibiotic resistance regulation, qRT-PCR experiments were carried out using the susceptible and less virulent A. baumannii A118 strain. We observed that, four out of five genes associated with β-lactam resistance as well as the fluoroquinolone resistance gene fusA were up-regulated (Supplementary Figure S1B).

To test the effect of H-NS on the antibiotic susceptibility profile of A. baumannii strains, disk diffusion and minimal inhibitory concentration (MIC) measurements were performed (Supplementary Table S3 and S4 and Supplementary Figure S2). Observing disk diffusion, an increase in the halo of 4 mm or more was observed for meropenem, imipenem, amikacin and gentamicin for both strains (Supplementary Table S3). MICs revealed 2-fold, 3-fold, 3-fold and 5-fold decreases in AB5075 Δhns for meropenem, imipenem, amikacin and gentamicin, respectively (Supplementary Table S4 and Supplementary Figure S2). In the A118 strain, a highly susceptible strain, slight changes in MICs values were observed in the Δhns strain for the carbapenems antibiotics (Supplementary Table S4). However, significant fold-decreases were observed for amikacin and gentamicin MICs (Supplementary Table S4).

Overall, an increase in expression of the analyzed antibiotic resistance genes is observed in Δhns mutant strains suggesting a pleiotropic modulation of gene expression. The increased expression of some genes cannot be correlated with the respective phenotype, such as the increase expression of aphA6 and aadB and the increase in susceptibility for amikacin and gentamicin, respectively. However, because antibiotic resistance mechanisms are multifactorial and other uncharacterized or unknown genes might be involved, H-NS could be partially responsible for the observed phenotypes.

A previous analysis using metallo-β-lactamases and DNA-damaging agents as stressors ⁹, showed that the AB5075 Δhns strain showed diminished ability to overcome stress ⁹. Moreover, imipenem MICs of strains expressing the different MBLs, showed a detrimental impact on the antibiotic resistance phenotype in the Δhns background ⁹.

The reduced MICs observed in the present work for carbapenems and aminoglycosides in the mutant background, could be related with the role of H-NS in stress response. In accordance with this result, RNA-seq analysis of the AB5075 mutant strain showed the up-regulation of genes that encode for proteins involved in counteraction stressful conditions (Supplementary Table S1).

In addition, the increased susceptibility for imipenem observed in both mutant strains can be explained in part by the significant increase in expression of carO (Supplementary Table S4, and Figure 1 and Supplementary Figure S1), since a correlation between the expression of CarO and the capacity of A. baumannii to allow the outer membrane permeation of imipenem was reported ^{14, 15}.

To further determine if changes at the transcriptional levels of efflux pumps could contribute to the altered antibiotic resistance phenotype in the Δhns mutant background, the transcriptomic data of AB5075 Δhns vs. AB5075 was examined. Our results showed that the expression of genes encoding AdeABC, AdeIJK as well as the efflux pump EmrAB were enhanced in AB5075 Δhns; however, the expression of genes that encode for AdeFGH were decreased (Figure 2A and Supplementary Table S2). qRT-PCR experiments further confirmed up-regulation of efflux pump genes in both A. baumannii Δhns mutant strains when compared with otherwise isogenic wild-type strains (Figure 2B and Supplementary S3). In trans complementation restored the expression levels for these genes in the AB5075 strain.

The up-regulation of efflux pumps can be related with the wide regulatory effects of H-NS causing transcriptional changes in genes related with A. baumannii’s adaptative response.

Diverse stress conditions, such as oxidative stress, mediated by global regulatory genes like SoxR, which is a global repressor protein involved in multidrug-resistance in Gram negative bacteria, cause the overexpression of efflux pumps contributing to multidrug-resistance phenotype ¹⁶. A recent in vitro study showed that under consecutive imipenem stress, A. baumannii evolved the ability to reduce susceptibility to various antimicrobials, including carbapenem, through overproduction of efflux pumps the RND super-family efflux pump ¹⁷. In other Gram-negative bacteria, as in Vibrio cholerae, it was shown that the loss of H-NS is involved in inducing an endogenous envelope/oxidative stress ¹⁸.

We observed a wide transcriptional response in the hns mutant, pointing out the central role of H-NS in modulating expression/repression of a wide variety of antibiotic resistance genes. Our results confirmed a pleiotropic role of H-NS as a transcriptional regulator that is playing a critical role in reprogramming the A. baumannii transcriptome related with antibiotic resistance and stress response.

Biofilm and Quorum sensing networks are also affected by H-NS

The transcriptomic comparison of A. baumannii AB5075 Dhns against the parental strain showed that expression of csu operon, which is essential for A. baumannii biofilm formation ¹⁹, was significantly down-regulated (Figure 3A and Supplementary Table S2). The differential expression of csuA/B and csuE was further confirmed to be down-regulated by qRT-PCR analysis in AB5075 Δhns (Figure 3B), while the AB5075 Δhns pMBLe-hns restored the wild-type level of expression of both csu genes analyzed. In addition, the changes in expression of these genes were correlated with the associated phenotype, since biofilm formation was significantly lower in AB5075 Δhns with respect to the parental strain (Figure 3 C). The rescued strain showed an increment of biofilm formation compared to the Δhns strain (Figure 3 C). Decreased biofilm formation was also seen in A118 Δhns and a decreased in the expression level of csuE was also observed in the mutant background (Supplementary Figure S4 A and B). Levels of expression of ompA, that encodes for the OmpA protein, partially involved in abiotic biofilm formation and known to be a virulence factor ²⁰, were up-regulated in both mutants strains (Figure 3B and Supplementary Figure S4 A).

It is known that in a biofilm the microorganisms reside and communicate with each other through quorum sensing mechanisms ²¹. AB5075 transcriptomic data analysis revealed that the expression of genes encoding enzymes that participate in acyl homoserine lactone (AHL) synthesis was up-regulated, as well as the expression of lactonase enzyme coding aidA gene ²² in the mutant strain (Figure 3A and Supplementary Table S2). The quorum network genes aidA, abaI, kar, and fadD were confirmed to be up-regulated in AB5075 Δhns mutant strains by qRT-PCR and the levels of expression were restored in hns complemented strain (Figure 3B). Moreover, the levels of expression of aidA and abaI genes were observed to be up-regulated in the susceptible model strain A118 (Supplementary Figure S4 A). In addition, we assessed the levels of AHL using Agrobacterium tumefaciens-based solid plate assays ^{23, 24}. The supernatants from cultures of AB5075 or AB5075 Δhns produced similar intensity of color, likely caused by an increase of both lactonase activity (quorum quenching) and AHL synthesis (quorum sensing) in the mutant strain (Figure 3D). The lower AHL production observed in the AB5075 Δhns pMBLe-hns can be explained by the decrease expression of genes associated with AHL synthesis (abaI, karD, and fadD) and the higher lactonase expression levels responsible of quorum quenching activity (aidA). Finally, AHL analysis for A118 Δhns showed lower intensity of blue color compared to A118 wild type strain (Supplementary Figure S4 C). This result suggested the production of a lactonase activity that is able to counteract the levels of large chain AHLs synthesized by this strain in the assayed conditions.

The genome-wide role of H-NS in A. baumannii was first studied in a hypermotile derivative of the reference strain ATCC 17978 ⁷. Transcriptomic analysis of the ATCC 17978 Δhns (strain 17978hm) identified 152 genes as DEGs (91 genes up-regulated and 61 down- regulated) when compared to the parental. The most striking up-regulation was observed in the quorum sensing genes encoding AbaI and the regulator AbaR. On the other hand, phenotypic and genetic analysis of biofilm coding genes showed no major differences between the wild type and mutant strains ⁷. Our transcriptomic and phenotypic data compared to that of ATCC 17978, demonstrated that H-NS regulon components might vary in a strain-specific manner consistent with the fact that different A. baumannii isolates can display significant variations in phenotypes known to be controlled by H-NS (i.e., cell surface hydrophobicity, adherence and motility) ²⁵.

Our present work further contributes to deciphering the role of the H-NS global regulator in the control of transcription of antibiotic resistance-related mechanisms.

Using distinct A. baumannii model strains, an extensive H-NS-controlled transcriptional response was observed. Our results reinforce the complexity of the regulatory network control by the transcriptional repressor H-NS that regulates the expression of genes involved in antibiotic resistance and persistence.

Overall, H-NS exerts a pleiotropic role in A. baumannii, influencing the expression of a wide variety of genes related to antibiotic resistance and stress response, suggesting that it is a good candidate to develop further alternative treatment approaches to combat A. baumannii infections.

Bacterial strains.

The multidrug and hypervirulent AB5075 strain and its isogenic h-ns mutant (AB5075 Dhns) were used in the present study. AB5075 Dhns was obtained from the A. baumannii AB5075 transposon mutant library ²⁶. In addition, to extend the role of H-NS on the response of A. baumannii, the model susceptible A118 and its isogenic mutant A118 Dhns were used. A. baumannii A118 Dhns was obtained as previously described ²⁷. Briefly, chromosomal DNA of AB5075 Dhns (hns::TET^R) was used to naturally transform the A118 strain and mutant cells were selected on Luria-Bertani (LB) agar plates supplemented with 30 µg/mL tetracycline (TET). A118 Δhns mutant strain was confirmed by automated DNA sequencing.

Electroporation

Electro-competent A. baumannii AB5075 Dhns cells were prepared and mixed with 25 ng of pMBLe-hns plasmid DNA (containing apramycin resistance) followed by electroporation with a Bio-Rad Gene Pulser instrument at 2.5 kV, 200 Ω, 25 µF. The electroporated cells were placed in recovery with 1ml of LB broth for 2 hours at 37 °C in a shaking incubator followed by culturing overnight at 37°C on LB agar containing 15 µg/ml apramycin. At least 10 colonies were picked to confirm the presence of the different plasmids. To confirm their presence, plasmid extraction followed by gel electrophoresis analysis

RNA extraction and RNA-seq analysis.

A. baumannii AB5075 and AB5075 Dhns cells were cultured in LB broth and incubated with agitation for 18 hours at 37 °C. Overnight cultures were then diluted 1:10 in fresh LB broth and incubated with agitation for 7 hours at 37 °C. The Direct-zol RNA Kit (Zymo Research, Irvine, CA, USA) was used to perform the RNA extraction in triplicates. RNA samples were DNase treated (Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instruction. Samples were confirmed to have no DNA contamination through PCR amplification of the 16S rDNA gene. RNA sequencing was outsourced to Novogene (Novogene Corporation, Sacramento, CA, USA). Ribosomal RNA-depletion was done using the Ribo-Zero kit (Illumina) and the construction of the cDNA library was performed with the TruSeq Stranded Total RNA Library Prep kit (Illumina) from three independent replicates per sample. Analysis of the quality of the illumina reads, trimming of low-quality bases and removal of Illumina adapters was performed as described previously ²⁸ . Reads were aligned to the genome of A. baumannii AB5075 using Burrows-Wheeler Alignment (BWA) software (v0.7.17) BWA and visualized using the Integrative Genomics Viewer (IGV). Read counts per gene were calculated using FeatureCounts ²⁹, and differential expression analysis was performed using DEseq2. Differentially expressed genes (DEGs) were defined as those displaying an FDR adjusted P-value of <0.05 and log2fold change > 1. RNA-seq data generated as a result of this work has been deposited to SRA with the accession GSE167117.

qRT-PCR was performed to confirm and analyze the expression with antibiotic associated genes. cDNA was prepared using the iScript™ Reverse Transcription Supermix for qRT-PCR (BioRad, Hercules, CA, USA) and quantitative PCR was performed using iQ™SYBR® Green Supermix (BioRad, Hercules, CA, USA) per the manufacturer’s recommendations, respectively. Results were analyzed using the 2^−ΔΔCt method in which recA ¹³ acted as the control gene. Experiments were performed in technical and biological triplicates. The results from experiments performed were statistical analyzed (t test) using GraphPad Prism (GraphPad software, San Diego, CA, USA). A P-value < 0.05 was considered significant.

Antimicrobial Susceptibility Assay

Antimicrobial commercial disks (BBL, Cockeysville, MD, USA) containing 10 μg of meropenem (MEM), 10 μg of ampicillin (AMP), 30 μg cefotaxime (CTX), 30 μg of ceftazidime (CAZ), 10 μg of imipenem (IMP), 15 μg of tigecycline (TIG), 30 μg of minocycline (MIN), 5 μg of ciprofloxacin (CIP), 10 μg of norfloxacin (NOR), 300 units of polymyxin B (PB), 30 μg of amikacin (AMK) and 10 μg of gentamicin (GN) were used. The inoculated plates were incubated at 37 °C for 18 hours. MICs against were performed using the reference methods agar dilution according to CLSI Standards ³⁰. Antimicrobial commercial E-strips (Liofilchem S.r.l., Italy) for meropenem (MEM), ampicillin (AMP), ceftazidime (CAZ), imipenem (IMP), ciprofloxacin (CIP), norfloxacin (NOR), amikacin (AN), and gentamicin (GN) were used. Mueller-Hinton agar plates were incubated at 37°C for 18 hours. CLSI breakpoints were used for interpretation ³⁰. The MIC for colistin susceptibility testing was determined using ComASP Kit^TM (Liofilchem^®) and following the manufacturer’s instructions.

Biofilm Assays

Biofilms assays were performed as described in previous work ¹³. A. baumannii cells were cultured in LB broth with agitation for 18 hours at 37ºC. Overnight cultures were centrifuged at 5,000 rpm at 4°C for 5 minutes. Pelleted cells were washed twice with 1X phosphate buffered saline (PBS) and then re-suspended in 1X PBS. The optical density at 600 nm (OD600) of each culture was then adjusted to 0.9–1.1 and the samples were the vortexed and diluted 1:100 in LB broth in technical triplicates, the inoculated 96-well polystyrene micro-titer platewas incubated at 37°C for 24 hours without agitation. The following day, the OD600 (ODG) was measured using a micro-plate reader (SpectraMaxM3 microplate/ cuvette reader with SoftMax Pro v6 software) to determine the total biomass. Wells were emptied using a vacuum pipette, washed three times with 1X PBS and stained with 1% crystal violet (CV) for 15 minutes. Excess CV was removed by washing three more times with 1X PBS and the biofilm associated with the CV was solubilized in ethanol acetate (80:20) for 30 m. The OD580 (ODB) was measured using a micro-plate reader and results were reported as the ratio of biofilm to total biomass (ODB/ODG). Experiments were performed in triplicates, with at least three technical replicates per biological replicate.

N-Acyl Homoserine Lactone (AHL) Detection

Agrobacterium tumefaciens-based solid plate assays were carried out to detect N-Acyl Homoserine Lactone (AHL) production ³¹ as described in previous work ²⁴. Initially, 500 µL of the homogenate were loaded in a central well of 0.7% LB agar plates supplemented with 40 µg of 5-bromo-3- indolyl-b-D-galactopyranoside (X-Gal) per mL and 250 µL (OD = 2.5) of the overnight culture of Agrobacterium tumefaciens biosensor. The presence of AHL was determined by the development of blue coloring ²³. As a positive control, 100 µL of N-Decanoyl-DL-homoserine lactone (C10-AHL) 12.5 mg/mL was utilized. Quantification of 5,5’-dibromo-4,4’-dichloro-indigo production in different conditions was determined using ImageJ software (NIH) by measuring the intensity of each complete plate, and subtracting the intensity measured in the negative control. The values were normalized to the positive control, which received the arbitrary value of 100.

Statistical Analysis.

Experiments performed at least in triplicates were statistically analyzed by ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism (GraphPad software, San Diego, CA, USA). A P-value < 0.05 was considered significant.

All procedures performed in this study were in accordance with the CSUF Institutional Biosafety Committee Approval plan (DBH117-01) and are in compliance with the NIH, CDC, OSHA and other environmental and occupational regulations.

Funding:

The authors’ work was supported by NIH SC3GM125556 to MSR, R01AI100560 to RAB, R01AI063517, R01AI072219 to RAB and 2R15 AI047115 to MET. CP and JE were supported by grant MHRT 2T37MD001368 from the National Institute on Minority Health and Health Disparities, National Institute of Health. DR has a MARC U*STAR fellowship by the National Institute of General Medical

Sciences of the National Institutes of Health under Award Number T34GM008612. This study was supported in part by funds and/or facilities provided by the Cleveland Department of Veterans Affairs, Award Number 1I01BX001974 to RAB from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development and the Geriatric Research Education and Clinical Center VISN 10 to RAB. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Department of Veterans Affairs. PICT 2017-1627 from the National Agency of Scientific and Technology Promotion to EGV. MRT and TS are recipient of a postdoctoral fellowship from CONICET. R.S. is a staff member from CONICET.

Author Contributions:

D.R., C.L., C.P., M.R.T., T.S., J.E., B.N., R.A.B, M.E.T. and M.S.R. conceived the study and designed the experiments. D.R., C.L., C.P., M.R.T., T.S., J.E., B.N., R.S., and M.S.R. performed the experiments and genomics and bioinformatics analyses. D.R., C.L., C.P., M.R.T., T.S., E.G.V., R.S., R.A.B., M.E.T. and M.S.R. analyzed the data and interpreted the results. R.A.B., M.E.T. and M.S.R. contributed reagents/materials/analysis tools. M.R.T., T.S., E.G.V., R.S., R.A.B., M.E.T. and M.S.R. wrote and revised the manuscript. All authors read and approved the final manuscript.

Conflicts of Interest:

The authors declare no conflict of interest.

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No competing interests reported.

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Histone-like Nucleoid-structuring Protein (H-NS) Regulatory Role in Antibiotic Resistance in Acinetobacter Baumannii

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Abstract

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Introduction

Results And Discussion

H-NS plays a role in modulating the expression of antibiotic resistance associated genes

Biofilm and Quorum sensing networks are also affected by H-NS

Conclusion

Materials And Methods

Bacterial strains.

Electroporation

RNA extraction and RNA-seq analysis.

Antimicrobial Susceptibility Assay

Biofilm Assays

N-Acyl Homoserine Lactone (AHL) Detection

Statistical Analysis.

Declarations

Funding:

Author Contributions:

Conflicts of Interest:

References

Additional Declarations

Supplementary Files

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Version 1