
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

72740
10.1038/s41598-024-72740-1
Article
Impact of AbaI mutation on virulence, biofilm development, and antibiotic susceptibility in Acinetobacter baumannii
Pumirat Pornpan 1
Santajit Sirijan 2
Tunyong Witawat 1
Kong-Ngoen Thida 1
Tandhavanant Sarunporn 13
Lohitthai Sanisa 1
Rungruengkitkun Amporn 1
Chantratita Narisara 1
Ampawong Sumate 4
Reamtong Onrapak 5
Indrawattana Nitaya nitaya.ind@mahidol.ac.th

67
1 https://ror.org/01znkr924 grid.10223.32 0000 0004 1937 0490 Department of Microbiology and Immunology, Mahidol University, Bangkok, Thailand
2 https://ror.org/04b69g067 grid.412867.e 0000 0001 0043 6347 Department of Medical Technology, School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat, Thailand
3 https://ror.org/058h74p94 grid.174567.6 0000 0000 8902 2273 Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan
4 https://ror.org/01znkr924 grid.10223.32 0000 0004 1937 0490 Department of Tropical Pathology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
5 https://ror.org/01znkr924 grid.10223.32 0000 0004 1937 0490 Department of Tropical Molecular Biology and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
6 https://ror.org/01znkr924 grid.10223.32 0000 0004 1937 0490 Biomedical Research Incubator Unit, Department of Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
7 grid.10223.32 0000 0004 1937 0490 Department of Research, Siriraj Center of Research Excellence in Allergy and Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
14 9 2024
14 9 2024
2024
14 2152117 5 2024
10 9 2024
© The Author(s) 2024
2024
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The quorum sensing (QS) system mediated by the abaI gene in Acinetobacter baumannii is crucial for various physiological and pathogenic processes. In this study, we constructed a stable markerless abaI knockout mutant (ΔabaI) strain using a pEXKm5-based allele replacement method to investigate the impact of abaI on A. baumannii. Proteomic analysis revealed significant alterations in protein expression between the wild type (WT) and ΔabaI mutant strains, particularly in proteins associated with membrane structure, antibiotic resistance, and virulence. Notably, the downregulation of key outer membrane proteins such as SurA, OmpA, OmpW, and BamA suggests potential vulnerabilities in outer membrane integrity, which correlate with structural abnormalities in the ΔabaI mutant strain, including irregular cell shapes and compromised membrane integrity, observed by scanning and transmission electron microscopy. Furthermore, diminished expression of regulatory proteins such as OmpR and GacA-GacS highlights the broader regulatory networks affected by abaI deletion. Functional assays revealed impaired biofilm formation and surface-associated motility in the mutant strain, indicative of altered colonization capabilities. Interestingly, the mutant showed a complex antibiotic susceptibility profile. While it demonstrated increased susceptibility to membrane-targeting antibiotics, its response to beta-lactams was more nuanced. Despite increased expression of metallo-beta-lactamase (MBL) superfamily proteins and DcaP-like protein, the mutant unexpectedly showed lower MICs for carbapenems (imipenem and meropenem) compared to the wild-type strain. This suggests that abaI deletion affects antibiotic susceptibility through multiple, potentially competing mechanisms. Further investigation is needed to fully elucidate the interplay between quorum sensing, antibiotic resistance genes, and overall antibiotic susceptibility in A. baumannii. Our findings underscore the multifaceted role of the abaI gene in modulating various cellular processes and highlight its significance in A. baumannii physiology, pathogenesis, and antibiotic resistance. Targeting the abaI QS system may offer novel therapeutic strategies for this clinically significant pathogen.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-72740-1.

Keywords

AbaI
Quorum sensing
Acinetobacter baumannii
Pathogenesis
Antibiotic resistance
Biofilm
Subject terms

Microbiology
Molecular biology
National Science Research and Innovation Fund (SRF)http://dx.doi.org/10.13039/501100004156 Mahidol University Fundamental Fund: fiscal year 2023 Indrawattana Nitaya http://dx.doi.org/10.13039/501100004704 National Research Council of Thailand N42A660376 Indrawattana Nitaya issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

The global rise of Acinetobacter baumannii as a critical pathogen in healthcare settings poses a significant challenge due to its remarkable adaptability and resistance to a wide range of antimicrobial agents. The persistence of A. baumannii is particularly concerning as it is facilitated by its ability to form biofilms and evade antibiotic treatment, making infections difficult to eradicate. Central to its pathogenesis is the quorum sensing (QS) system (AbaI/AbaR), which enables coordinated behavior among bacterial populations, facilitating the establishment of infections and adaptation to host environments. Among the genes involved in the QS system, abaI plays a pivotal role, with its product being crucial for the synthesis of the signaling molecule N-acylhomoserine lactone (AHL), thereby modulating the bacterial behavior and pathogenicity1,2. Beyond QS, A. baumannii employs a various resistance mechanism that includes altering membrane permeability and producing enzymes that degrade antibiotics. Proteomic studies have shed light on the role of specific proteins related to the outer membrane and efflux systems, which are upregulated in response to antibiotic stress, contributing significantly to the multidrug-resistant (MDR) phenotype of A. baumannii3.

The outer membrane of Gram-negative bacteria serves as a barrier that protects the cell from harmful substances, including antibiotics. By altering the permeability of this membrane, A. baumannii can control what substances are allowed to pass through, thereby limiting the effectiveness of antibiotics4. Outer membrane proteins (OMPs) are integral components of the outer membrane in Gram-negative bacteria, playing crucial roles in various cellular processes including transport, signaling, and structural integrity. Among the diverse array of OMPs, SurA, OmpA, OmpW, and BamA stand out as key players, each contributing uniquely to the biology and physiology of the bacterial cell5,6. By altering the permeability of their membrane, A. baumannii can control what substances are allowed to pass through, thereby limiting the effectiveness of antibiotics4. Moreover, the regulatory proteins OmpR and GacA-GacS play crucial roles in orchestrating gene expression and coordinating various cellular processes in bacteria. This system modulates the expression of downstream target genes involved in various cellular processes such as virulence, biofilm formation, motility, and secondary metabolite production. It is also associated with the transition from planktonic to biofilm lifestyles and plays a central role in bacterial adaptation and pathogenicity7. Biofilm formation confers protection against host immune responses and antimicrobial agents, making infections difficult to eradicate and leading to chronic or recurrent infections8.

Understanding these complex resistance mechanisms, alongside the impact of QS, is crucial for developing effective therapeutic strategies to combat A. baumannii infections. In addressing these challenges, this study focuses on elucidating the influence of the abaI gene on A. baumannii phenotype, encompassing biofilm formation, cell morphology, motility, and antibiotic susceptibility. By employing targeted mutagenesis to delete the abaI gene and creating a complemented strain, we aim to dissect the role of this QS gene in the bacterial pathogenicity. The study integrates proteomic analysis, biofilm quantification, microscopic examination of cell shape, motility assays, and antibiotic susceptibility testing to provide a comprehensive overview of how abaI impacts A. baumannii behavior and resistance profiles. This approach not only enhances our understanding of A. baumannii pathogenic mechanisms but also identifies potential targets for novel antimicrobial strategies.

Results and discussions

Construction of a ΔabaI knockout mutant and a complemented strain of A. baumannii

To investigate the effect of abaI QS system in the A. baumannii, we constructed a stable markerless abaI knockout mutant (ΔabaI) using a pEXKm5-based allele replacement method9. The joined flanking sequence on both sides of abaI was subcloned into pEXKm5 to delete the entire abaI gene from the chromosome of A. baumannii ATCC 17978. The absence of abaI was validated by PCR and DNA sequencing (Fig. S1). Both A. baumannii ATCC 17978 WT and the ΔabaI knockout mutant underwent proteomic analysis. Additionally, the influence of abaI QS system in the A. baumannii growth was investigated through the optical density of the culture over time. The growth of the mutant and complement strains did not differ from that of parental strain (Fig. 1).

Fig. 1 Growth character of A. baumannii strains used in this study. WT (A. baumannii ATCC17978), ΔabaI, and ΔabaI::abaI strains were grown in LB broth at 37 °C with shaking. OD was determined at 600 nm. The data points and error bars represent the mean ± SEM from triplicate experiments.

Proteomic analysis

Proteomic assay was approached to identify protein expression among A. baumannii WT and ΔabaI mutant strains. Protein profiling was performed in triplicate, and only the proteins detected in all replicates at a confidence level of 95% were subsequently identified. Then, significantly (> 2-fold change) differentially expressed proteins (DEPs) were determined in A. baumannii ΔabaI mutant strain compared with WT strain. Pathway analysis of these proteins was performed using the Uniprot database. From a total of 990 proteins detected in A. baumannii ΔabaI mutant strain, there were 54 upregulated and 198 downregulated DEPs (Table S1). Differentially Expressed Genes (DEGs) were classified based on their Gene Ontology (GO) functional terms using Uniprot database (Table S2). Top twenty proteins in biological process were presented in Fig. 2. Exclude from unknown group, most of downregulated proteins were grouped in translational process, followed by in proteolysis mechanism, and in DNA templated transcription. We also identified the upregulated and downregulated DEPs of mutant strain (ΔabaI) which related to membrane protein, membrane transporter, membrane formation and antibiotic resistance (Table 1). Among the membrane proteins, we found the reduced expression of PPIase SurA (A0A829KEQ5), which is important protein involved in the biogenesis of outer membrane proteins (OMPs) in Gram-negative bacteria like A. baumannii. SurA assists in the biogenesis of newly synthesized OMPs by facilitating their folding into stable conformations and guiding them to the β-barrel assembly machinery (BAM) complex for insertion into the outer membrane. In the absence of SurA or under conditions of SurA depletion, the folding and assembly of OMPs can be compromised, leading to outer membrane defects and increased susceptibility to environmental stresses, including antibiotic treatment10. Besides, the virulence factor group presented down regulation level of a major component of OMPs i.e., OmpA family protein (A0A335N5J1), OmpW family protein (Outer membrane beta-barrel protein, A0A0H4V0P0), and BamA (an outer membrane β-barrel assembly protein, A0A062IKF0). OmpA is one of the most abundant outer membrane proteins in Gram-negative bacteria, including A. baumannii. It plays several roles, including maintaining cell shape, participating in bacterial adhesion to host cells and surfaces, and contributing to the integrity of the outer membrane11. OmpA mutations in A. baumannii, can compromise outer membrane integrity, potentially reducing virulence and altering antibiotic susceptibility, underscoring its significance in pathogenesis and antimicrobial resistance12. OmpW, a small outer membrane protein, functions primarily as a porin. It forms an eight-stranded β-barrel structure within the outer membrane, facilitating the transport of small hydrophobic molecules across the membrane13. The downregulation of OmpW may lead to decreased permeability for certain compounds. This reduction in permeability can potentially decrease susceptibility to some antibiotics by preventing the compounds from reaching their intracellular targets. Its downregulation could impair the ability of bacteria to sense and respond to changes in their population density or to environmental cues, potentially reducing their virulence as reported in the study of Cronobacter sakazakii14. BamA is a key component of the BAM complex, which facilitates the insertion and folding of OMPs. It serves as a scaffold for OMP insertion and is essential for outer membrane integrity15. Downregulation of bamA resulted in significant defects in outer membrane stability and exhibited reduced virulence in both in vitro and in vivo models of infection16.

Fig. 2 A. baumannii WT (A. baumannii ATCC17978) and ΔabaI mutant proteomic analysis: DEPs ontology groups. A number of genes displaying > 2-fold alteration in wild-type compared to ΔabaI-mutant cells by gene ontology.

Table 1 Protein related to membrane protein, membrane formation and antibiotic resistance.

Uniprot accession	Subcellular localization	Protein name	Classification	
A0A829KEQ5	Outer membrane-bounded periplasmic space [GO:0030288]	Chaperone SurA (Peptidyl-prolyl cis-trans isomerase SurA) (PPIase SurA) (EC 5.2.1.8)	Downregulation	
A0A335N5J1	Cell outer membrane [GO:0009279]	OmpA family protein	Downregulation	
A0A0H4V0P0	Outer membrane [GO:0019867]	OmpW family protein (Outer membrane beta-barrel protein)	Downregulation	
A0A062IKF0	Cell outer membrane [GO:0009279]	Outer membrane protein assembly factor BamA	Downregulation	
A0A009GXE2		Transcriptional regulatory protein ompR	Downregulation	
A0A829KFI6	Membrane [GO:0016020]	LemA family protein	Downregulation	
A0A009NJC5	Cytoplasm [GO:0005737]	Protein-export protein SecB	Downregulation	
A0A009H796	Membrane [GO:0016020]	Porin B	Downregulation	
A0A833PIS0	Cytoplasm [GO:0005737]	Cell shape-determining protein MreB	Downregulation	
A0A829KFI6	Membrane [GO:0016020]	LemA family protein	Downregulation	
A0A429MLG1	Gram-negative-bacterium-type cell wall [GO:0009276]; plasma membrane	General secretion pathway protein GspL	Upregulation	
A0A009GI99		Metallo-beta-lactamase superfamily protein	Upregulation	
A0A090B7T5		DcaP-like protein	Upregulation	

Furthermore, this study identified a diminished expression of OmpR (A0A009GXE2), the transcription factor and corresponding response regulator for the membrane-associated sensor kinase EnvZ, in the mutant strain (ΔabaI). Aside from the regulation of OMPs in response to osmotic signals, OmpR and EnvZ act as global regulators controlling the expression of many genes such as flagellar biosynthesis and motility in Escherichia coli17. Homologs of the OmpR/EnvZ also control virulence in several other pathogens, including Salmonella enterica serovar Typhimurium, and Yersinia pestis18,19. Although A. baumannii does not encode flagella, A. baumannii strain AB5075 exhibit significant differences in the surface motility on soft agar plates. Mutation of ompR in A. baumannii strain AB5075 reduced motility significantly in the opaque variant compared to the wild-type strain1,20. Therefore, the reduction of OmpR expression in A. baumannii ΔabaI mutant strains might possibly affect to their motility when compared with WT strains.

In the mutant strain (ΔabaI), a significant decrease in the expression of the sensor element GacA-GacS (LemA) was observed. The GacS/GacA two-component system, which is important in the plant pathogen Pseudomonas syringae pv. syringae, is required for lesion formation, swarming, and the production of proteases and N-acyl-l-homoserine lactone compounds involved in quorum sensing22. Similar systems, such as the RcsB-RcsC system, control capsule biosynthesis in E. coli23, and gacS-like genes are present in A. baumannii24. When GacS/GacA is downregulated, it causes multiple defects, including reduced motility, and biofilm formation22. Our findings are consistent with these reports, suggesting that the observed downregulation in the mutant strain may similarly impact these physiological traits. SecB, a chaperone protein, binds and maintains newly made preproteins in an unfolded state. It then targets these preprotein substrates to the SecA-SecYEG translocase in the inner membrane, enabling their translocation across the membrane in a translocation-competent conformation25. In this study, there was a noted decrease in the expression of SecB (A0A009NJC5) potentially affecting protein export in a mutant strain. Downregulation secB results in significantly reduced translocation efficiency of SecB-dependent substrates like maltose binding protein (MalE) and OmpA. Consequently, secB mutant strains exhibit pleiotropic defects such as hypersensitivity to membrane stresses, and reduced virulence as documented in a study on E. coli and A. baumannii25,26. Porin B (A0A009H796), refer to Omp33-36 protein, is an outer membrane protein that has been linked to antibiotic resistance and virulence in A. baumannii. The role of Omp33-36 extends to facilitating the bacterial adherence, invasion, and cytotoxicity, as well as contributing to its survival and persistence within the host27. We found downregulated porin B in mutant strain which may resulting in the permeability reduction of the bacterial cell envelope to antibiotics, potentially making the bacteria more susceptible to these compounds27,28.

In A. baumannii and related species, the cell shape-determining protein MreB (A0A833PIS0) plays a crucial role in defining cell morphology through the orchestration of peptidoglycan synthesis, which is a key component of the bacterial cell wall29. A malfunction or reduction in MreB activity, as observed in the mutant strain, can lead to a loss of the characteristic rod shape, compromising the cell wall strength and leading to cell lysis, a finding supported by several studies30–32.

However, in the mutant strain (ΔabaI), the DEPs that showed increased levels of expression included the General Secretion Pathway protein (GspL, A0A429MLG1), metallo-beta-lactamase (MBL) superfamily proteins (A0A009GI99), and the DcaP-like protein (A0A090B7T5). The GspL protein, a component of the Type II Secretion System (T2SS), is prevalent among many Gram-negative bacteria and facilitates the transport of proteins from the periplasm through the outer membrane into the extracellular space. It is vital for the secretion of diverse enzymes and toxins, thereby playing a crucial role in bacterial pathogenicity and interactions with the environment33. Despite the upregulation of MBL superfamily proteins in the mutant strain, which would typically confer resistance to carbapenems—a class of broad-spectrum antibiotics often used as a last resort for treating multidrug-resistant bacterial infections—the observed decrease in imipenem and meropenem MICs suggests the involvement of other mechanisms. These could include alterations in outer membrane permeability, efflux pump activity, or other regulatory pathways34. Similarly, the upregulation of the DcaP-like protein (A0A009GI99) in Acinetobacter, particularly in the mutant strain, could significantly impact antibiotic resistance and virulence. The structural similarity between the DcaP-like protein and AbOmpA implies that the DcaP-like protein may play a role in maintaining outer membrane integrity in bacteria. This combination of factors highlights the complexity of antibiotic resistance mechanisms and suggests that multiple pathways may contribute to the observed phenotypes3,35,36.

Several studies have investigated the influence of the quorum sensing system, including abaI, abaR, and abaM, on the pathogenicity of A. baumannii, examining various aspects such as growth, motility, virulence, biofilm formation, and antibiotic susceptibility37–42. For instance, Sun et al. demonstrated that the abaI/abaR quorum sensing system significantly impacts biofilm formation and motility by downregulating genes associated with virulence factors and motility, leading to reduced virulence and impaired biofilm formation in abaI deletion strains. Similarly, Tang et al. reported that the deletion of abaI increased susceptibility to specific antibiotics, underscoring its role in antibiotic resistance38. Furthermore, transcriptomic analysis by Xiong et al. indicated that abaI deletion leads to significant downregulation of genes involved in energy production and conversion39. This study corroborates these findings by showing that abaI deletion in A. buamannii results in decrease motility, reduced virulence, and increased susceptibility to antibiotics, similar to previous studies37. Additionally, our research provides a novel and detailed proteomic profile of A. baumannii ΔabaI, revealing the specific roles of functional proteins in the bacterium physiological processes. By integrating proteomic and phenotypic analyses, we uncovered correlations between altered protein expression and phenotypic changes, such as compromised membrane integrity, impaired biofilm formation, and reduced virulence. This comprehensive approach not only elucidates the impact of abaI deletion on a molecular level but also identifies potential new pathways and mechanisms that contribute to A. baumannii pathogenicity and antibiotic resistance. Our findings offer deeper insights into the role of abaI in the virulence mechanisms of A. baumannii, advancing the current understanding by highlighting novel functional proteins and their specific contributions to the bacterium’s adaptation and survival. This integrative analysis paves the way for future research to explore these newly identified pathways and their potential as targets for therapeutic intervention.

Mutant showed differences in biofilm formation and morphology

To determine the impact of deletion of abaI gene on biofilm formation, and cell morphology, Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) were used in this study. In Fig. 3a, SEM images revealed a dense, multi-layered biofilm structure in WT and complemented (ΔabaI::abaI) strains. In contrast, mutant strain (ΔabaI) showed a significant reduction in density, with large areas devoid of biofilm cells. WT and complement biofilms exhibited a highly organized structure, with cells closely packed in a uniform matrix. Mutant biofilms displayed disrupted arrangements, with cells scattered and less cohesion observed. The extracellular matrix of WT and complement appeared as a thick, continuous layer surrounding the cells.

Fig. 3 Biofilm morphology of A. baumannii wild type (WT), ΔabaI mutant, and ΔabaI::abaI complemented strains analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). (a) SEM images showing the biofilm structure formed by WT, ΔabaI, and ΔabaI::abaI strains. Biofilms were visualized to assess surface coverage and three-dimensional architecture. (b) TEM images of negatively stained biofilms illustrating the cellular morphology and extracellular matrix of the different strains. WT denotes wild type; ΔabaI indicates the abaI gene knockout; ΔabaI::abaI represents the abaI knockout strain complemented with the abaI gene. Images are representative of three independent experiments.

This study conducted a detailed morphological analysis WT, mutant strain (ΔabaI), and the complemented (ΔabaI::abaI) strains of A. baumannii using TEM. The focus was on assessing alterations in cell shape, membrane structure, and cell edges. WT strain exhibited uniform cell shapes with smooth, intact membranes and well-defined edges (Fig. 3b). Mutant strains (ΔabaI) showed considerable deviation in cell shape, with irregularities and bulging observed. Membrane integrity was compromised in several areas, leading to blurred or indistinct cell edges. Complemented strains (ΔabaI::abaI) largely restored the WT morphological features, displaying more uniform cell shapes and improved membrane integrity. The cell edges were clearer and more defined compared to the mutant strain, although slight variations from the WT were noted. The mutant strain altered morphology suggests that the abaI gene deletion impacts cell wall synthesis or stability, leading to irregular shapes and compromised membranes.

Further, the level of biofilm formation of WT, mutant (ΔabaI), and complemented (ΔabaI::abaI) bacterial strains were assessed through crystal violet staining, a conventional method for measuring biofilm mass (Fig. S2). The statistical analysis of the differences between groups revealed that mutant (ΔabaI) groups showed biofilm formation ability significantly lower than WT (p-value < 0.05). Moreover, the comparison between the WT and complemented (ΔabaI::abaI) strains revealed no significant difference in biofilm formation between these two groups (p-value = 0.977). These results imply that the deletion of the abaI gene significantly affects biofilm formation compared to the WT, whereas the complemented strain (ΔabaI::abaI) does not significantly differ from the WT, indicating a restoration of the biofilm formation capability which is in accordance with the previous finding42,43.

Mutant showed difference surface-associated motility

In our investigation of the impact of abaI gene on the motility of A. baumannii, we conducted a series of motility assays comparing the WT, mutant (ΔabaI), and complemented (ΔabaI::abaI) strains. The assays revealed no differences in swimming and swarming motilities among the WT, ΔabaI mutant, and ΔabaI::abaI complemented strains (Fig. 4a,b). However, in twitching motility assays, the WT strain exhibited pronounced activity at the agar interface, while the twitching zone of the ΔabaI mutant was significantly reduced (Fig. 4c). In contrast, the complemented (ΔabaI::abaI) strain demonstrated the increase twitching activity when compared to the mutant strain. These findings indicate that the abaI gene plays a role in regulating twitching motility in A. baumannii, which influences the bacterial ability to colonize and spread across surfaces. This aligns with findings from other studies, suggesting a broader regulatory role for abaI in bacterial motility44.

Fig. 4 Surface motility assays depicting the swimming (a), swarming (b), and twitching (c) abilities of A. baumannii wild type (WT), ΔabaI mutant, and ΔabaI::abaI complemented strains. (a) Swimming motility was assessed by measuring the diameter of the circular zone of bacterial migration from the inoculation point on a semisolid agar plate. (b) Swarming motility was evaluated by observing the expansion of bacterial growth on the surface of a nutrient-rich agar medium. (c) Twitching motility was examined by visualizing the formation of spreading zones underneath the agar surface caused by bacterial movement. WT denotes wild type; ΔabaI indicates the abaI gene knockout; ΔabaI::abaI represents the abaI knockout strain complemented with the abaI gene. Data are representative of three independent experiments.

Mutation lessens virulent in Galleria model

To evaluate the role of the abaI gene in the pathogenic capabilities of A. baumannii, we utilized G. mellonella larvae, carefully selected for uniformity in size and health, and organized them into distinct groups for infection trials with the WT, mutant (ΔabaI), and complemented (ΔabaI::abaI) strains. Our observations revealed that the WT strain exhibited noticeable virulence, evidenced by a mere 50% survival rate among the larvae after 48 h (Fig. 5). Conversely, larvae infected with the mutant strain (ΔabaI) showed no fatalities, implying a loss of pathogenicity attributable to the absence of the abaI gene, thereby marking a 100% survival rate. Intriguingly, reintroducing the abaI gene into the mutant strain through complementation (ΔabaI::abaI) resulted in a partial revival of the bacterial virulence, as indicated by a reduced survival rate of 70%, approaching that of the WT strain. These findings indicate the impact of the abaI gene in the virulence of A. baumannii. The complemented (ΔabaI::abaI) strain restoring its virulent phenotype to a degree, further solidifies the specific contribution of the abaI gene to the pathogenicity of A. baumannii. This study not only advances our comprehension of the genetic underpinnings of A. baumannii pathogenicity but also paves the way for the development of targeted therapies against this formidable pathogen.

Fig. 5 Percentage survival of A. baumannii wild type (WT), ΔabaI mutant, ΔabaI::abaI complemented strains, and phosphate-buffered saline (PBS) control. Bacterial cultures were subjected to a stressor or treatment, followed by assessment of survival rates. Survival percentages were calculated relative to the initial bacterial population. WT denotes wild type; ΔabaI indicates the abaI gene knockout; ΔabaI::abaI represents the abaI knockout strain complemented with the abaI gene. Data represent the mean of three independent experiments with error bars indicating standard deviations.

Enhanced antibiotic susceptibility in mutant

The impact of a mutation in the abaI gene on the antibiotic resistance profile of A. baumannii was investigated by comparing WT strains to mutant (ΔabaI) and complemented (ΔabaI::abaI) strains. The results revealed that the ΔabaI mutant strain has increased susceptibility to imipenem, meropenem, gentamicin, kanamycin, tetracycline, and vancomycin, compared to its WT counterparts (Table 2). Interestingly, the minimum inhibitory concentrations (MICs) for antibiotics such as ampicillin, piperacillin/tazobactam, and cefoperazone/sulbactam were notably higher in the ΔabaI mutant strain compared to the WT. In contrast to the WT strain, the ΔabaI mutant exhibited increased susceptibility to antibiotics targeting the cell membrane. However, the mutant strains displayed higher minimum inhibitory concentrations (MICs) for the beta-lactam antibiotics ampicillin and cefoperazone/sulbactam compared to the wild type. Despite the upregulation of metallo-beta-lactamase (MBL) superfamily proteins and DcaP-like protein, the MICs for carbapenem antibiotics, such as imipenem and meropenem, were unexpectedly lower in the mutant strain than in the wild type (0.19 vs. 0.25). This lower MIC could be attributed to other compensatory mechanisms within the mutant strain, such as alterations in membrane permeability or efflux pump activity, which may enhance carbapenem uptake or reduce resistance, offsetting the effect of MBL upregulation. These results suggest a more complex relationship between MBL expression and carbapenem resistance in the ΔabaI mutant strains. These observations suggest that changes in the abaI gene play a significant role in the observed increase in antibiotic resistance, hinting at additional mechanisms that require further exploration.

Table 2 Minimal inhibitory concentration of antibiotics used in this study.

Antibiotic	MIC (µg/ml)	
WT	∆abaI	∆abaI::abaI	
Penicillin G	> 32	> 32	> 32	
Ampicillin	24	32	24	
Ceftazidime	8	8	6	
Cefepime	1.5	1.5	1.5	
Piperacillin/tazobactam	3	4	2	
Cefoperazone/sulbactam	0.75	1.0	0.75	
Imipenem	0.25	0.19	0.25	
Meropenem	0.25	0.19	0.25	
Gentamicin	0.75	0.38	0.25	
Kanamycin	1.0	0.75	1.0	
Spectinomycin	32	32	32	
Streptomycin	12	12	12	
Tetracycline	3	1.5	1.5	
Vancomycin	128	64	128	

Materials and methods

Bacterial strains, plasmids, and culture

A. baumannii ATCC 17978 was purchased from American Type Culture Collection (ATCC) and was used as the WT strain. Escherichia coli strain DH5α and RHO3 were used for cloning and generation of A. baumannii mutant strains. Bacteria were grown in Luria Burtani medium (LB; Difco Laboratory, Sparks, MD, USA) at 37 °C with shaking. Kanamycin (50 µg/ml) was added to the growth medium to maintain plasmid in E. coli. A. baumannii merodiploids were selected on medium supplemented with kanamycin (500 µg/ml) and ampicillin (100 µg/ml).

Construction of A. baumannii ΔabaI mutant and complementary strain

The A. baumannii abaI mutant and complementary strains were constructed using markerless allele replacement method9 with some modifications45,46. To delete the abaI gene, a DNA fragment consisting of 483 bp upstream and 649 bp downstream regions, derived from GenBank (locus_tag = “KIP77_RS01625”) of A. baumannii, was synthesized and cloned into the pUC57 vector (GenScript, Jiangsu, China). Subsequently, the fragment was cut through digestion with NotI and EcoRI (New England Biolabs, MA, USA), followed by ligation to the pEXKm5 vector using the same restriction enzymes. The pEXKm5 plasmid containing the fragment was transformed into E. coli RHO3 and mobilized into A. baumannii ATCC 17978 using the conjugation method. The obtained conjugants were selected on LB agar containing 500 µg/mL of kanamycin and incubated for 24–48 h at 37 °C. To resolve the merodiploid, the bacteria were grown in yeast extract-tryptone agar containing 15% (w/v) sucrose at room temperature. The isolated colonies were screened for kanamycin-sensitive clones. The abaI deletion mutants were validated by PCR using primers flanking the deleted alleles, F-RS01625 and R-RS01625 (Table 3). Additionally, the absence of the pEXKm5 plasmid was confirmed using oriT primers (Table 3).

Table 3 Oligonucleotide sequences in this study.

Primer names	Sequence (5′−3′)	Purpose	Size	Source	
F- RS01625	5′-gcggccgcAGCATGGTTAGCATACCCCC-3′	Amplification of full length abaI	1699	This study	
R- RS01625	5′-gaattcCGCTAGCTTACTCCACCACAC-3′	
OriT-F	5′-TCCGCTCATAACCCTGCTTC-3′	Validation of the presence of pEXKm5 plasmid backbone	236	Lopez, C.M., et al., 2009	
OriT-R	5′-CAGCCTCGCAGAGCAGGATTC-3′	

To facilitate gene complementation, a similar pEXKm5-based allele exchange method was utilized. The full-length sequence of A. baumannii ATCC 17978 abaI was amplified from the genomic DNA of A. baumannii ATCC 17978 using the F1-RS01625 and R2-RS01625 primers (Table 3). Then, the DNA product was subjected to NotI and EcoRI digestion and subsequently ligated with the NotI and EcoRI -digested pEXKm5 vector. Similar to the generation of the deletion mutant, the pEXKm5 containing the whole sequence of abaI was transformed into E. coli RHO3 for conjugation into the A. baumannii abaI mutant. The complementation of abaI was screened by PCR using primers flanking the deleted alleles, F-RS01625 and R-RS01625 (Table 3) and verified by DNA sequencing.

Bacterial growth assay

A single isolated colony of A. baumannii was inoculated in LB broth and incubated at 37 °C with shaking at 200 rpm for 24 h. Then, the overnight-culture of bacteria was washed with phosphate buffered saline (PBS) and adjusted to an optical density (OD) at 600 nm (OD600) of 0.5. To examine the growth kinetics, the prepared A. baumannii was added into fresh LB medium at a ratio of 1:500 and incubated at 37 °C with shaking at 200 rpm. OD600 was measured at predetermined time points.

Label-free proteomic analysis

To explore the protein expression profiles, proteins were harvested through centrifugation, followed by ice-cold acetone precipitation (1:5 v/v). Following precipitation, the protein pellet was reconstituted in a solution containing 0.25% RapidGest SF (Waters, Manchester, UK) in 15 mM ammonium bicarbonate (Sigma-Aldrich, St. Louis, MO). The total protein (60 µg) underwent gel-free digestion. Subsequently, sulfhydryl bond reduction was conducted using 5 mM DTT (Sigma-Aldrich, St. Louis, MO) in 15 mM ammonium bicarbonate at 72 °C for 1 h. Following the reduction, sulfhydryl alkylation was carried out using IAA (Sigma-Aldrich, St. Louis, MO) in 15 mM ammonium bicarbonate at room temperature for 30 min in the dark. The solution was cleaned up using a Zeba Spin Desalting Column (Thermo Scientific Co., IL, USA). The flow-through solution was enzymatically digested by trypsin (Promega Co., Madison, WI, USA) at a ratio of 1:50 (enzyme–protein) and incubated at 37 °C for 3 h. Subsequently, the digested solution was dried and reconstituted in 0.1% formic acid before being subjected to LC–MS/MS. The experiment was conducted in three-biological replications (n = 3).

The LC-MS/MS spectrum data were acquired in the positive mode using an HF-X Hybrid Quadrupole-Orbitrap™ Mass Spectrometer, coupled with an EASY-nLC1000 nano-LC system equipped with a nano C18 column. The mobile phase A comprised 0.1% formic acid in water, while mobile phase B consisted of 90% acetonitrile with 0.1% formic acid. The samples were directly loaded onto an analytical C18 column. Separation was achieved using a linear gradient of 2–45% mobile phase B at a constant flow rate of 300 nL/min for a duration of 135 min. The analytical column was regenerated with 90% mobile phase B for 10 min and re-equilibrated with 5% mobile phase B for 35 min. Peptide analysis was performed utilizing a data-dependent (TopN15) acquisition method, followed by higher-energy collisional dissociation (collision energy = 28 eV). Full scan mass spectra were acquired at an m/z ratio of 400 to 1600 with an AGC target set at 3 × 106 ions and a resolution of 120 k. MS/MS scanning was initiated when the automatic gain control target reached 1 × 105 ions and a resolution of 15 k. The raw mass spectra (.raw file) were processed using MaxQuant_v2.3.1.0 software and matched against the UniProt protein database (https://www.uniprot.org/; organism: Acinetobacter baumannii). Protein identification and quantification were carried out with the following parameters: MS tolerance, 20 ppm; MS/MS tolerance, 0.05 Da; digestion enzyme, trypsin; fixed modification, cysteine carbamidomethylation; and variable modification, methionine oxidation. The false discovery rate (FDR) for the identification of peptides and proteins was set at 1%. Differentially Expressed Genes (DEGs) were classified based on their Gene Ontology (GO) functional terms by Uniprot database.

Motility assays

Motility assays were carried out as previously described40. In brief, approximately 106 CFU of A. baumannii were cultured overnight on nutrient agar plates. The motility was analyzed on 0.5% agar plates supplemented with 5 g/L tryptone and 2.5 g/L NaCl. To reduce variation between plates, 30 mL of medium was poured into each plate in a laminar flow hood and left for 1 h with the lids off, before being used immediately. A 1 µL drop of freshly grown culture (OD600 ~ 0.6) was then placed at the center of each plate to evaluate surface-associated motility. For twitching motility41, the overnight culture was picked with a toothpick and stabbed into the interface between the agar bottom and the polystyrene Petri dish, interphase. For each bacterial strain, assays were performed at least three times.

Biofilm production capability and cell morphology

To assessed crystal violet biofilm assay, colonies of A. baumannii were dissolved in phosphate-buffered saline, pH 7.4 (PBS), and turbidity of the solutions were adjusted to 0.5 McFarland standard. Twenty microliters of the prepared cells were added to a 96-well microtiter plate containing 180 µL of LB broth and incubated at 37 °C for 24 h, then the fluids were drained. The wells were washed with 200 µL of PBS, pH 7.4, stained the cells with 1% crystal violet, and incubated at 37 °C for 30 min. The cell culture wells were washed with water and allowed to dry at room temperature. The biofilm produced by the bacteria was determined by destaining the stained crystal violet color in the culture well by adding with 200 µL 33% acetic acid. The absorbance of the resulting solution was measured at absorbance 595 nm using a microplate spectrophotometer. The test was repeated five times.

The scanning electron microscope (SEM) technique was employed to determine the biofilm formation ability of A. baumannii strains and cell morphology. Briefly, 200 µL of bacteria from each strain were placed into a 24-well plate containing the SEM blocking film. The plate was then incubated at 37 °C overnight to allow the bacteria to form a biofilm. Subsequently, the well was washed twice with PBS, fixed with 2.5% glutaraldehyde in 0.1 M sucrose phosphate buffer (SPB), pH 7.4, at 4 °C overnight, washed with SPB three times for 10 min each, and postfixed with 1% osmium tetroxide (OsO4) in 0.1 M SPB. Following this, all culture wells were washed with SPB three times for 10 min each, dehydrated with ethanol, and dried using a critical point drying machine (Leica; model EM CPD300, Germany) before coating with a 10 nm thick gold–palladium film (Quorum; model Q150R-S Plus, UK). The prepared samples were then examined for biofilm formation and cell morphology using SEM (Jeol; model JSM-6610LV, Japan).

Additionally, transmission electron microscopy (TEM) was performed to examine bacterial ultrastructure. A. baumannii was inoculated in LB broth at 37 °C with aeration overnight. One mL of each culture was then centrifuged (3000 ×g, 25 °C, 5 min). The cell sludge was resuspended in 2.5% glutaraldehyde in 0.1 M SPB, pH 7.4, at 4 °C overnight, and the cells were washed with SPB three times for 10 min each. The cell sludge was then fixed again in 1% OsO4 in 0.1 M SPB, washed in 0.1 M SPB, stained with 2% uranyl-acetate, and imaged by TEM (Hitachi; model HT7700, Japan).

Galleria mellonella infection assay

G. mellonella caterpillar was used as a model for A. baumannii infection to study the bacterial toxicity/virulence. Forty larvae were used in this experiment. All were 2–2.5 cm in length, 250–300 mg in body weight, and free of melanization. After 18 h of growth, A. baumannii was diluted to a concentration of ∼ 3 × 108 CFUs/mL in PBS by adjusting the OD600. A Hamilton syringe was used to inject 105 CFU of the bacterial suspension into the body cavity of G. mellonella larvae via the proleg. Each control larva was injected with PBS. Following injection, larvae were incubated in the dark at 37 °C for 5 days and checked daily for viability. The day after post-injection, larvae were individually investigated for pigmentation and mobility. Larvae were considered dead when they displayed no movement in response to gentle prodding with a pipette tip. The numbers of dead larvae and times of death were recorded, and the survival graph was plotted.

Antimicrobial sensitivity assays

Minimum inhibitory concentration (MIC) based on Epsilometer method (E-test) was used to determine the antimicrobial sensitivity of A. baumannii. Antimicrobial agents in this study included Penicillin G, Ampicilin, Ceftazidime, Cefepime, Piiperacillin/tazobactam, Cefoperazone//sulbactam, Imipenem, Meropenem, Gentamicin, Kanamycin, Spectinomycin, Streptomycin, Tetracycline, and Vancomycin (Liofilchem, Roseto Degli Abruzzi, Italy). The antimicrobial susceptibility protocols and interpretation were followed the guidelines of the Institute for Clinical and Laboratory Standards version 202047.

Statistical analysis

The results are presented as the mean values and standard deviations derived from three independent experiments. Statistical comparisons between control and test groups were performed using one-way ANOVA followed by Tukey’s post-hoc test, using GraphPad Prism 5 software (La Jolla, CA, USA). Significance was determined at a threshold of p < 0.05.

Supplementary Information

Supplementary Material 1

Supplementary Material 2

Supplementary Material 3

Supplementary Material 4

Acknowledgements

This research project work is supported by the Mahidol University (Fundamental Fund: fiscal year 2023 by National Science Research and Innovation Fund (SRF)) and funded by National Research Council of Thailand (NRCT) (N42A660376) and Mahidol University. Prof. Herbert Schweizer, Norther Arizona University provided the pEXKm5 plasmid and he is acknowledged with thanks.

Author contributions

P.P., S.S. and N.I. contributed to the conception and design of the study. P.P., W.T., T.K., S.T., S.L., A.R., O.R., S.A., S.S., and N.I. performed the experiments. N.C. and N.I. provided the reagents and technical guidance on the experiments. P.P., S.S. and N.I. analyzed the data and wrote the manuscript. P.P., S.S. and N.I. edited the manuscript. All authors read and approved the final version of the manuscript.

Data availability

All the mass spectrometry raw data have been deposited in the Science Data Bank repository, accession number 10.57760/sciencedb.08749 (https://www.scidb.cn/en/anonymous/SXpFRmZp).

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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