BaeR and H-NS control CRISPR-Cas-mediated immunity and virulence in Acinetobacter baumannii

Abstract

Acinetobacter baumannii balances its remarkable ability to acquire antibiotic resistance genes via horizontal gene transfer (HGT) with the immune defense functions of its CRISPR-Cas system, forming a dynamic equilibrium governed by intricate transcriptional regulation. However, the regulatory mechanisms underlying the I-Fb CRISPR-Cas system in A. baumannii remain poorly understood. This study elucidated a multitiered regulatory axis mediated by BaeR and H-NS that coordinates immune defense and virulence expression in the I-Fb CRISPR-Cas system. Using DNA pull-down and electrophoretic mobility shift assay (EMSA), we demonstrated that H-NS directly binds AT-rich regions within the cas3 promoter, suppressing both interference activity and adaptive immunity of the I-Fb CRISPR-Cas system. Intriguingly, the two-component regulator BaeR controlled this suppression by positively regulating H-NS expression. The results revealed that Δcas3 mutants exhibited increased biofilm thickness, elevated the extracellular matrix component poly N-acetyl glucosamine (PNAG) production, upregulated pilus expression, and significantly enhanced epithelial cell adhesion. Strikingly, Δh-ns-cas3 and ΔbaeR-cas3 double-knockout strains showed no statistically significant differences in virulence phenotypes compared to the Δcas3 single mutants. These findings indicate CRISPR-Cas-mediated inhibition of biofilm formation is abolished upon cas3 deletion, thereby releasing the regulatory constraints imposed by BaeR and H-NS. This dysregulation leads to excessive biofilm and extracellular matrix component accumulation, ultimately amplifying bacterial colonization capacity and pathogenicity in host environments. This discovery reveals the dual regulatory roles of BaeR and H-NS in the A. baumannii I-Fb CRISPR-Cas system, mediating both immune defense and virulence modulation. These insights establish a theoretical foundation for novel antimicrobial strategies targeting CRISPR-Cas regulatory networks.IMPORTANCEA. baumannii, a leading cause of drug-resistant nosocomial infections, evolves antibiotic resistance through horizontal gene transfer (HGT) while employing CRISPR-Cas systems to limit foreign DNA invasion. This study reveals that the I-Fb CRISPR-Cas system, typically a defense mechanism, functions as a repressor of virulence traits in A. baumannii. We demonstrate that the transcriptional regulators H-NS and BaeR form a hierarchical axis suppressing Cas3 expression, thereby constraining biofilm formation and host adhesion. Strikingly, CRISPR-Cas deficiency enhances virulence, thickens biofilms, elevates PNAG production, and enhances epithelial colonization through escape from BaeR-/H-NS-mediated control. This work redefines CRISPR-Cas as a dual-function module balancing immune defense and pathogenicity, exposing the BaeR-H-NS-Cas3 axis as a druggable target for novel anti-infectives aimed at disrupting bacterial adaptive evolution.

Keywords: Acinetobacter baumannii; BaeR; CRISPR-Cas; H-NS; biofilm; immunity; virulence.

Fig 1

Identification of the cas3 promoters and the binding site of H-NS activating the cas3 promoter. (A) The reporter plasmids containing empty (AB43-C) and three different lengths of the promoter (163 bp, 297 bp, and 425 bp) were constructed. The activities of putative promoters of cas3 were measured by fluorescence emission. (B) Silver staining analyzed the proteins bound to the cas3 promoter. 1, 2: two independent experiments. Input: The positive control group is full protein without the probe. DPD: Experimental group is the eluted protein with biotin labeling. NC: negative control group is the eluted protein without biotin labeling. The arrow represents specific bands. (C) PCR and double enzyme digestion verification of constructed strains. M: the 5,000 bp DNA marker, 1: PCR products of h-ns, 2: double enzyme digestion verification. (D) SDS‐PAGE analysis of rH-NS after induction with 1 mM IPTG for 6 h at 37°C. M: the 180 kDa protein marker, C: pET30a without IPTG induction, S: soluble proteins, I: insoluble proteins. (E) Western blot analysis of rH-NS with anti-His-tag monoclonal antibody (lane 1). M: the protein marker (F) EMSAs for rH-NS binding to the promoter of cas3. (G) EMSAs of subfragments P1, P2, P3, P4, and P5 with purified rH-NS. (H) DNase I footprinting analysis of H-NS binding to the Cas3 promoter region. The experiments were repeated three times. Error bars show mean ± SD. ***P < 0.001—one-way ANOVA with Dunnett post hoc tests (A).

Fig 2

The binding sequences of BaeR activating Cas3 expression. (A) PCR and double-enzyme digestion verification of constructed strains. (B) SDS‐PAGE analysis of rBaeR after induction with 1 mM IPTG at 37°C for 6 h. M: the 180 kDa protein marker, C: pET30a without IPTG induction, S: soluble proteins, I: insoluble proteins. (C) Western blot analysis of rBaeR using an anti‐His mouse monoclonal antibody (lane 1). (D) EMSAs for rBaeR binding to the promoter of cas3. (E) DNase I footprinting analysis of rBaeR binding to the cas3 promoter region. (F) EMSAs of subfragments P1, P2, P3, P4, and P5 with purified rBaeR.

Fig 3

Competitive EMSAs and BaeR increase the H-NS expression. (A, B) Competitive EMSAs for binding of rH-NS or rBaeR to the promoters of cas3. (C) Western blot analysis of H-NS or BaeR expression in mutation strains. (D) qRT-PCR analysis of gene h-ns or baeR expression in mutation strains. (E) EMSAs for rBaeR binding to the promoter of h-ns. The experiments were repeated three independent times. Data represent mean ± SD. ns P > 0.05, **P < 0.01, and ***P < 0.0001—one-way ANOVA with Tukey’s post hoc test (C and D).

Fig 4

H-NS represses the activity of CRISPR-Cas interference and spacer acquisition. (A) The cas3 expression was measured by qRT-PCR. (B) The type I-Fb CRISPR-Cas locus in AB43. A schematic of the experiments utilized a nontargeted plasmid, three CRISPR-targeted plasmids, and three CRISPR-mutation plasmids. (C) Retention of the control plasmid and the CRISPR-targeted plasmid in WT and mutants. (D) The transformation efficiency of WT and mutants was quantified as the percentage transformation by the CRISPR-targeted plasmid compared with that of the parent vector lacking the targeted sequence. (E) Each of the strains harbored the CRISPR-mutation plasmid to promote adaptation. Each adaptation event results in the acquisition of a new spacer and CRISPR repeat. PCR of single colonies analyzed the integration of new CRISPR spacers into the CRISPR locus. Each adaptation event results in the acquisition of a new spacer and CRISPR repeat, which is exhibited by an expansion of the CRISPR locus. Error bars denote the mean ± SD from n = 3 replicates. ns P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001—one-way ANOVA with Tukey’s (A) and Dunnett’s (C and D) post hoc test.

Fig 5

BaeR represses the activity of CRISPR-Cas interference and spacer acquisition. (A) Retention of the CRISPR-targeted plasmid. (B) Transformation efficiency of CRISPR-targeted plasmids. (C) Acquisition of new spacer sequences analyzed by PCR. Error bars represent SD from n = 3 replicates. ns P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001—one-way ANOVA with Dunnett’s (A and B) post hoc test.

Fig 6

The expression of extracellular matrix components and biofilm formation. (A) Measurement of biofilm biomass by crystal violet staining. Data are representative of three independent experiments; bar graphs show mean ± SD. ***P < 0.001—one-way ANOVA with Dunnett’s post hoc test. (B through H) Light microscopic images of biofilms formed in mutation strains. (I) Measurement of extracellular matrix components. Poly N-acetyl glucosamine (PNAG) is a known extracellular matrix component of the hydrophobic biofilm of A. baumannii, and the lectin wheat germ agglutinin (WGA) binds selectively to PNAG. Error bars represent SD from n = 3 replicates. ***P < 0.001—one-way ANOVA with Dunnett’s post hoc test. (J through P) Confocal laser microscopic images of biofilms formed by mutation strains on the surface of a 24-well chamber glass slide after 24 h.

Fig 7

Evaluation of virulence and expression of Csu pili. (A) The survival of Galleria mellonella (n = 10) infected with AB43 and mutants. Survival analyses were performed using Kaplan-Meier survival curves. (B, C) The colonization of bacteria into the lungs or BALF of mice was sacrificed after 24 h of intranasal infection and measured by CFU counting of bacterial colonies on LB agar plates. Each experiment was performed with 6 mice. The qRT-PCR analysis of gene fimD (D), csuAB (E), and pilA (F) expressions. (G, H) Adherence and invasiveness of AB43 and mutants to epithelial cells A549. The experiments were repeated three independent times. Error bars show mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001—one-way ANOVA with Dunnett’s post hoc test.