CRISPR-Cas in Acinetobacter baumannii Contributes to Antibiotic Susceptibility by Targeting Endogenous AbaI

Yuhang Wang; Jie Yang; Xiaoli Sun; Mengying Li; Pengyu Zhang; Zhongtian Zhu; Hongmei Jiao; Tingting Guo; Guocai Li

doi:10.1128/spectrum.00829-22

. 2022 Aug 8;10(4):e00829-22. doi: 10.1128/spectrum.00829-22

CRISPR-Cas in Acinetobacter baumannii Contributes to Antibiotic Susceptibility by Targeting Endogenous AbaI

Yuhang Wang ^a,^b, Jie Yang ^a, Xiaoli Sun ^b, Mengying Li ^a,^c, Pengyu Zhang ^a,^b, Zhongtian Zhu ^a,^d, Hongmei Jiao ^a,^d, Tingting Guo ^a,^b,^c, Guocai Li ^a,^b,^c,^d,^✉

Editor: Monica Adriana Garcia-Solache^e

Reviewed by: Jiaqi Cheng^f

PMCID: PMC9430643 PMID: 35938813

ABSTRACT

Acinetobacter baumannii is a well-known human opportunistic pathogen in nosocomial infections, and the emergence of multidrug-resistant Acinetobacter baumannii has become a complex problem for clinical anti-infective treatments. The ways this organism obtains multidrug resistance phenotype include horizontal gene transfer and other mechanisms, such as altered targets, decreased permeability, increased enzyme production, overexpression of efflux pumps, metabolic changes, and biofilm formation. A CRISPR-Cas system generally consists of a CRISPR array and one or more operons of cas genes, which can restrict horizontal gene transfer in bacteria. Nevertheless, it is unclear how CRISPR-Cas systems regulate antibiotic resistance in Acinetobacter baumannii. Thus, we sought to assess how CRISPR-Cas affects biofilm formation, membrane permeability, efflux pump, reactive oxygen species, and quorum sensing to clarify further the mechanism of CRISPR-Cas regulation of Acinetobacter baumannii antibiotic resistance. In the clinical isolate AB43, which has a complete I-Fb CRISPR-Cas system, we discovered that the Cas3 nuclease of this type I-F CRISPR-Cas system regulates Acinetobacter baumannii quorum sensing and has a unique function in changing drug resistance. As a result of quorum sensing, synthase abaI is reduced, allowing efflux pumps to decrease, biofilm formation to become weaker, reactive oxygen species to generate, and drug resistance to decrease in response to CRISPR-Cas activity. These observations suggest that the CRISPR-Cas system targeting endogenous abaI may boost bacterial antibiotic sensitivity.

IMPORTANCE CRISPR-Cas systems are vital for genome editing, bacterial virulence, and antibiotic resistance. How CRISPR-Cas systems regulate antibiotic resistance in Acinetobacter baumannii is almost wholly unknown. In this study, we reveal that the quorum sensing regulator abaI mRNA was a primary target of the I-Fb CRISPR-Cas system and the cleavage activity of Cas3 was the most critical factor in regulating abaI mRNA degradation. These results advance our understanding of how CRISPR-Cas systems inhibit drug resistance. However, the mechanism of endogenous targeting of abaI by CRISPR-Cas needs to be further explored.

KEYWORDS: Acinetobacter baumannii, CRISPR-Cas, abaI, antibiotic susceptibility

INTRODUCTION

Acinetobacter baumannii (A. baumannii) is a lactose-nonfermenting, Gram-negative bacterium that can survive in natural environments and hospitals because it has little need for living conditions (1). In 2017, the World Health Organization (WHO) listed carbapenem-resistant A. baumannii as a significant priority, implying that new antibiotics are urgently needed to combat this species (2). Overuse of antibiotics has caused the emergence of multidrug resistance in A. baumannii, as a consequence of either horizontal gene transfer (3) or other factors, such as altered targets, decreased membrane permeability (4), increased production of degrading enzymes (5, 6), overexpression of efflux pumps (7), metabolic changes (8), biofilm formation (9, 10), or increased nutrient sequestration mechanisms (11).

The CRISPR-Cas system is an acquired intrinsic immune defense system of bacteria that maintains the stability of the bacterial genome by resisting the invasion of exogenous genetic material such as plasmids, phages, etc. (12). A total of 39% of sequenced bacterial and 88% of archaeal genomes contain CRISPR-Cas systems (13), with approximately 70% of pathogenic bacteria containing type I CRISPR-Cas systems (14). Based on the composition of Cas proteins, CRISPR-Cas systems are currently classified into two main categories, six types (I–VI), and more than 30 subtypes (15, 16). Type I-F is the most prevalent CRISPR-Cas system in A. baumannii (17). The I-Fb CRISPR-Cas system consists of Cas1, Cas3, Csy1, Csy2, Csy3, Csy4, and CRISPR arrays (18, 19). Studies have revealed that Cas3 cleaves invading RNA (20) by acting as a single-strand DNA nuclease and an ATP-dependent helicase (21). This cascade is composed of Csy1 to Csy4 proteins, wherein Csy4 processes crRNA transcripts, and the Csy1 to Csy3 proteins are necessary to stabilize the crRNA generated by Csy4 (22).

In addition to being an essential part of the prokaryotic immune system that prevents viral infection, the CRISPR-Cas systems also have various roles in physiology, such as boosting bacterial virulence and countering antibiotic resistance (23 –25). For example, recent studies have shown that A. baumannii uses the I-Fb CRISPR-Cas system to stop the spread of antibiotic resistance genes (26). Our recent research also demonstrated that the I-Fb CRISPR-Cas-related gene Csy1 in AB43 was upregulated when treated with most antibiotics and only downregulated when treated with doxycycline and kanamycin as an antibiotic pressure (27). However, it is unclear why this phenomenon occurs and its specific mechanism. CRISPR-Cas also incorporates cues for cell population density into its regulation.

Using quorum sensing (QS), bacteria can communicate with each other through extracellular signals, allowing them to obtain information about their surroundings, densities, and metabolic activities (28). In A. baumannii, AbaR (QS receptor) and AbaI (QS synthase), through a self-secreted signaling molecule called N-Acyl homoserine lactone (AHL), control the expression of specific phenotypes such as motility, antibiotic resistance, survival, and biofilm formation (29, 30). More specifically, QS activates type I-F CRISPR-Cas expression and CRISPR adaptation in P. aeruginosa, allowing CRISPR-Cas activity to increase in tandem with bacterial cell density (31). When bacterial populations have a high cell density and a high chance of phage infection and dissemination, this method guarantees maximal CRISPR-Cas activity to exert its immune defense. On the other hand, when bacteria invade mammalian host cells, the Cas3 of P. aeruginosa recognizes and cleaves the QS regulator lasR mRNA to enhance its virulence (32). The details of how CRISPR-Cas systems work to regulate antibiotic resistance remain unknown.

We, therefore, investigated the role of the Type I-Fb CRISPR-Cas system in A. baumannii in modulating QS operation as it affects bacterial resistance to antibiotics. We found that the Type I-Fb CRISPR-Cas of A. baumannii degraded mRNA of the QS master regulator abaI, and the cleavage activity of Cas3 was the most critical factor in regulating abaI mRNA degradation. Our study shows how CRISPR-Cas systems mechanistically regulate antibiotic resistance in A. baumannii.

RESULTS

Detection of CRISPR-Cas systems in A. baumannii isolates.

Of the 245 randomly collected A. baumannii clinical isolates, no isolate was susceptible to all 24 antibiotics (Fig. 1A). Among the 245 A. baumannii isolates tested, 16/245 (6.53%) and 20/245 (8.16%) isolates were resistant to only one or two of the nine classes of antibiotics tested, respectively. Specifically, 209/245 (85.31%) were classified as multidrug-resistant (MDR; resistance to three or more classes of antibiotics). In A. baumannii, most identified CRISPR-Cas systems were type I-F, and the Cas operon was composed of six to seven genes (17) (Fig. 1B). We screened for the CRISPR-Cas systems using PCR in the collected 245 A. baumannii clinical isolates. Of the 64 isolates with the I-Fa CRISPR-Cas system, only one isolate (resistant to one category of antibiotics tested) had a complete I-Fa CRISPR-Cas system, while the other 63 (49 MDR) I-Fa CRISPR-Cas-positive clinical isolates were incomplete. Similarly, of 49 isolates with the I-Fb CRISPR-Cas system, only one isolate (resistant to one category of antibiotics tested) was found to have a complete I-Fb CRISPR-Cas system. The other 48 (40 MDR) I-Fb CRISPR-Cas positive clinical isolates were incomplete. MLST of the 113 CRISPR-Cas-positive isolates revealed 40 different sequence types (STs). The most prevalent ST was ST1145 (25/113, 22.12%), followed by ST195 (15/113, 13.27%), ST1696 (12/113, 10.62%), and ST1417 (9/113, 7.96%) (Table S3). Furthermore, only these four STs have both I-Fa and I-Fb CRISPR-Cas systems, and no crossover distribution has been found in other STs. Additionally, 132 isolates were not positive for a CRISPR-Cas system, 4/132 (3.03%) and 8/132 (6.06%) isolates were resistant to only one or two of the nine classes of antibiotics tested, respectively, and 120/132 (90.91%) were classified as MDR (Fig. 1C).

The PCR results showed that among these 245 strains, most strains with incomplete or without CRISPR-Cas systems were MDR (Fig. 1D). To explore whether drug resistance in A. baumannii possessing an incomplete CRISPR-Cas system is associated with a specific Cas protein, we statistically analyzed the relationship between drug resistance phenotypes and cas genes in these 113 CRISPR-Cas-positive strains, and the results are shown in Table 1. We found that all cas gene-negative strains had significantly higher resistance rates than positive strains. I-Fa csy3-negative or I-Fb cas3-negative had the highest resistance rates in I-Fa and I-Fb cas gene-negative strains, respectively. In this regard, it is speculated that the incomplete CRISPR-Cas system, especially the loss of I-Fa csy3 and I-Fb cas3, may affect antibiotic resistance in A. baumannii. The I-Fb CRISPR-Cas system is highly conserved and can prevent the horizontal transfer of junction elements (33); we identified a complete I-Fb CRISPR-Cas system in AB43. This strain resisted one category of antibiotics tested (Table 2). Thus, we wanted to explore the critical cas gene that inhibits drug resistance and the possible mechanisms in the I-Fb CRISPR-Cas system.

TABLE 1.

Relationship between the cas genes and the drug resistance phenotype in 245 clinical A. baumannii strains^a

Cas	Positive		Negative
Cas	No. of MDR isolates	% (n = 245)	No. of MDR isolates	% (n = 245)
I-Fa-cas1	49	20%	160	65.31%
I-Fa-cas3	7	2.86%	202	82.45%
I-Fa-csy2	22	8.98%	187	76.33%
I-Fa-csy3	1	0.41%	208	84.9%
I-Fa-cas6	28	11.43%	181	73.88%
I-Fb-cas1	40	16.33%	169	68.98%
I-Fb-cas3	5	2.04%	204	83.27%
I-Fb-csy1	14	5.71%	195	79.59%
I-Fb-csy2	9	3.67%	200	81.63%
I-Fb-csy3	8	3.27%	201	82.04%
I-Fb-csy4	34	13.88%	175	71.43%

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MDR, multidrug resistance (resistance to three or more classes of antibiotics).

TABLE 2.

Drug resistance of AB43-derived strains^a

Antimicrobial	Drug	AB43	AB43Δcrispr-cas	AB43ΔabaI	AB43Δcrispr-cas-abaI
Penicillin	Piperacillin	R	R	I	I
β-lactam/β-lactamase inhibitor combinations	Ampicillin-sulbactam	S	R	S	S
	Piperacillin-tazobactam	S	R	S	S
	Ticarcillin-clavulanate	S	R	S	S
Cephems	Cefotaxime	I	R	S	S
	Ceftazidime	S	R	S	S
	Ceftriaxone	I	R	S	S
	Cefepime	S	R	S	S
Carbapenems	Imipenem	S	R	S	S
Carbapenems	Meropenem	S	R	S	S
Aminoglycosides	Amikacin	S	R	S	S
	Gentamicin	S	R	S	S
	Tobramycin	S	R	S	S
Fluoroquinolones	Ciprofloxacin	S	R	S	S
	Levofloxacin	S	R	S	S
	Gatifloxacin	S	I	S	S
Lipopeptides	Polymyxin B	S	S	S	S
Tetracyclines	Tetracycline	S	R	S	S
	Doxycycline	S	R	S	S
	Minocycline	S	S	S	S
Folate pathway inhibitors	Sulfisoxazole	S	R	S	S
	Roxithromycin	S	R	S	S
	Rifampicin	S	R	S	S
	Chloramphenicol	S	R	S	S

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R, resistance; I, intermediate; S, sensitivity. Boldface indicates antimicrobial susceptibility results change.

Complete CRISPR-Cas system represses antibiotic resistance in A. baumannii.

We first constructed one mutant by deleting the CRISPR-Cas cluster (AB43Δcrispr-cas) and restored the locus in a deletion mutant background (AB43Δcrispr-cas/pcrispr-cas). Compared to the wild type (WT), the deletion of the crispr-cas and its restoration did not affect A. baumannii growth (Fig. S1). We further constructed a series of single-locus mutants derived from AB43, including cas1, cas3, csy1, csy2, csy3, csy4, and crispr, as well as their corresponding complemented strains. Antimicrobial susceptibility tests for the WT, deletion mutants, and complemented strains were evaluated using 24 drugs belonging to nine major antibiotic types. While the AB43 and all the gene rescue mutants were susceptible to most antibiotics, deletion of any component or total abolishment of the CRISPR-Cas system rendered AB43 significantly resistant to most tested drugs (Table 2 and Table S5–S11).

Transcriptomic analysis of the entire CRISPR-Cas knockout mutant.

After demonstrating that the entire CRISPR-Cas system was required to suppress antibiotic resistance in A. baumannii, we sought to clarify the molecular mechanisms of this phenomenon. To address this issue, we performed transcriptomic analyses of AB43 and AB43Δcrispr-cas. As shown in Fig. 2A, and a total of 1,403 genes showed a significant difference in expression between these two strains, with 189 genes being upregulated and 1,214 genes being downregulated in the AB43Δcrispr-cas strain. KEGG enrichment analysis revealed that these genes were related to the two-component system (TCS) and various bacterial metabolism-related pathways (Fig. 2C and D). Specifically, these genes with repressed expression were correlated with the TCS, while those with increased expression were involved in multidrug efflux pumps (Fig. 2B). Particularly, genes associated with the ATP-binding cassette (ABC) transporters and multidrug efflux pumps increased dramatically, implying more of this essential function was present in the AB43Δcrispr-cas strain.

FIG 2 — Transcriptomic analysis of an entire *crispr-cas* knockout mutant. (A) Volcano plot annotations of differentially expressed genes (DEGs) in a AB43Δ*crispr-cas* strain. The x- and y axes in indicate changes in expression and statistically significant degrees, respectively. The adjusted P-value (P < 0.05; Student's t test with Benjamini–Hochberg false discovery rate adjustment) and |log2Fold change|≥1 were applied for determining DEG significance. (B) Selected differential expression genes involved in two-component systems and multidrug efflux pumps. An analysis of KEGG enrichment gene expression of (C) upregulated DEGs; and (D) downregulated DEGs. Data from three biological replicates were used.

CRISPR-Cas inhibits A. baumannii efflux pumps.

To further validate the CRISPR-Cas regulation of efflux pumps, we next monitored the efflux activity in different AB43-derived genotypes using ethidium bromide (EtBr) as a fluorescent probe. The results showed that increased efflux of EtBr occurred in deletion mutants (Fig. 3A, Fig. S2A). Similarly, qRT-PCR results showed that the mRNA expression of genes related to the efflux pump, such as ABC transporters (macB and emrB) (34), the major facilitator superfamily (MFS) (craA, rpoB, tetB, and abaQ), the resistance-nodulation-cell division (RND) superfamily (adeB and adeG), and the small multidrug resistance (SMR) protein family (abeS and abeM) in the knockout strain were elevated (Fig. 3B and C) (35). The efflux pump superfamilies use energy from the proton motive force (PMF), except for the ABC superfamilies, which utilize energy from ATP hydrolysis to facilitate the efflux of substances within a cell (36). We used the pH-sensitive fluorescent probe BCECF-AM to evaluate the PMF of these strains. AB43 deletion mutants increased fluorescence compared to a WT strain (Fig. 3D, Fig. S2B). As the PMF drives ATP synthesis (37), the intracellular levels of ATP also significantly increased in deletion strains (Fig. 3E, Fig. S2C). Altogether, these results showed that the entire CRISPR-Cas system significantly represses the mRNA expression of ABC transporters and efflux pump genes and inhibits the energy required for efflux.

FIG 3 — CRISPR-Cas inhibits *A. baumannii* efflux pumps. (A) Bacteria were coincubated with EtBr (8 μg/mL final concentration) or the known efflux pump inhibitor CCCP (10⁻⁴ M) at 37°C. Then, EtBr efflux from cells was monitored with an excitation wavelength of 530 nm and an emission wavelength of 600 nm for 60 min. (B and C) RNA was isolated when AB43, AB43Δ*crispr-cas*, and the complementary strain AB43Δ*crispr-cas*/pcrispr-cas grew to an OD₆₀₀ of 1.0 in liquid LB medium. Transcripts of indicated efflux pump-related genes (B) ABC transporters (*macB*, *emrB*) and MFS (*craA*, *rpoB*, *tetB*, and *abaQ*); (C) RND (*adeB* and *adeG*) and SMR (*abeS* and *abeM*) in AB43Δ*crispr-cas* strains were then quantified by qRT-PCR. (D) CRISPR-Cas decreased PMF based on the fluorescence intensity of BCECF-AM-probed *A. baumannii* cells. (E) A luciferin-luciferase bioluminescence assay has decreased the production of intracellular ATP in AB43. Significance was evaluated using nonparametric one-way ANOVA (**, P < 0.01; ***, P < 0.001; NS, not significant). All data are presented as the mean ± standard error of the mean (SEM).

CRISPR-Cas deletion impacts A. baumannii biofilm formation and membrane permeability.

Next, we explored other resistance mechanisms besides efflux pump activity. As biofilm formation is a well-known mechanism for antibiotic resistance in A. baumannii (38), we used crystal violet to detect the deletion strains’ biofilm formation. Our results showed that AB43Δcrispr-cas formed a significantly more robust biofilm than WT or its complemented strains (Fig. 4A). Similarly, AB43Δcas1, AB43Δcas3, AB43Δcsy1, AB43Δcsy2, AB43Δcsy3, AB43Δcsy4, and AB43Δcrispr all developed higher levels of bacterial biofilms than their respective gene complementary strains (Fig. S3A).

FIG 4 — *Crispr-cas* deletion impact on *A. baumannii* biofilm formation and membrane permeability. (A) Measurement of AB43Δ*crispr-cas* biofilm biomass by crystal violet staining. *A. baumannii* strains were cultured in sterile 96-well microtiter plates, and the absorbance was recorded at 570 nm. (B) *Crispr-cas* deletion reduced the membrane permeability of *A. baumannii*. (C) *Crispr-cas* deletion reduced the outer membrane permeability of *A. baumannii*. (D) *Crispr-cas* deletion dissipates membrane potential. DiSC3(5) dye was applied to determine the membrane potential. The dissipated membrane potential of *A. baumannii* was measured with an excitation wavelength of 622 nm and an emission wavelength of 670 nm. Mean values of three independent experiments and SEM values are shown. *, P < 0.05; ***, P < 0.001.

Since multidrug resistance in A. baumannii often requires an inner membrane permease (39), we hypothesized that the CRISPR-Cas system might inhibit the membrane permeability of AB43. To test this, we used the fluorescent probe propidium iodide (PI) to test membrane permeability (40), and we found that the deletion mutants tested exhibited significantly lower membrane permeabilities than the WT strain and complementary strains (Fig. 4B, Fig. S3B). We also assessed the effects of CRISPR-Cas on the permeability of the outer membrane (OM) via fluorescence intensity analysis. N-Phenyl-1-naphthylamine (NPN), a hydrophobic fluorescent probe that releases fluorescence when interacting with the hydrophobic parts of a phospholipid bilayer (41), was used to assess the permeability of the OM. In line with previous membrane permeability results, the knockout of any CRISPR-Cas component decreased the OM permeability of AB43 (Fig. 4C, Fig. S3C). DiSC3(5) was exploited to evaluate the bacterial membrane potential. We found this significantly reduced deletion mutants compared to AB43 and the complemented strain (Fig. 4D, Fig. S3D). These results indicated that dysfunction of the CRISPR-Cas system could enhance AB43 biofilm biomass and dampen bacterial membrane permeability, which shows a synergistic effect with efflux pumps.

CRISPR-Cas contributes to reactive oxygen species generation in A. baumannii.

It was recently reported that the production of reactive oxygen species (ROS) is essential in the sensitivity of A. baumannii to antibiotics (42). To that end, we wanted to explore whether ROS was involved in the process of CRISPR-Cas inhibition of drug resistance. The results showed that the deletion strains had reduced generation of total ROS (Fig. 5A, Fig. S4A) and increased intracellular superoxide dismutase (SOD) activity compared with AB43 (Fig. 5B, Fig. S4B). In cells, ROS included hydrogen peroxide (H₂O₂), superoxide (O2·−), and hydroxyl radicals (OH·). It was also interesting to note that CRISPR-Cas significantly promoted the production of H₂O₂ (Fig. 5C, Fig. S4C). Moreover, inhibiting tricarboxylic acid (TCA) cycle activity reduced ROS formation, and this TCA cycle activity required NAD. To further determine whether CRISPR-Cas impacts the TCA cycle, the NAD⁺/NADH ratio in deletion strains was determined. Consistent with the results above, the deletion strains had significantly increased NAD⁺/NADH ratios, indicating a reduced TCA cycle in mutants (Fig. 5D, Fig. S4D). We concluded that the CRISPR-Cas system increases ROS damage by promoting the TCA cycle and reducing the activity of the bacterial antioxidant system.

FIG 5 — CRISPR-Cas contributes to reactive oxygen species generation in *A. baumannii*. (A) Deleting *crispr-cas* drastically reduced total ROS. *A. baumannii* strains were probed with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). (B) CRISPR-Cas impairs bacterial oxidative defenses. A biochemical assay measured SOD activity in cells. (C) CRISPR-Cas induces the production of H₂O₂. (D) An accelerated TCA cycle was observed in AB43 or complement strains. (A–C) P-values (*, P < 0.05; ***, P < 0.001) were determined by unpaired t test between two groups or one-way ANOVA between multiple groups, respectively. All data are presented as the mean ± SEM.

CRISPR-Cas represses drug resistance by targeting abaI mRNA.

Next, we sought to determine which transcripts or genes were being disabled by CRISPR-Cas upstream of these different phenotypic changes. Previous studies have suggested that the I-F CRISPR-Cas system in P. aeruginosa can target the bacterial quorum-sensing regulator lasR mRNA and reduce bacterial virulence (32). Moreover, the QS system regulates bacterial luminescence, toxin production, disinfectants tolerance, motility, biofilm formation, spore formation, and drug resistance (43). Intriguingly, we found that the QS synthase gene abaI contains one region matching the CRISPR array. The region from nucleotides (nt) 29 to 39 in abaI is partly matched with spacer 101 and repeats (Fig. 6A). Thus, we utilized a specific bacterial biosensor (44), the A. tumafaciens KYC55 strain, to determine the amount of AHL activity in AB43-derived strains. As shown in Fig. 6B, the AHL activity of AB43Δcrispr-cas was stronger than the WT strain. Compared with AB43, more than half (5/8, 62.5%) of the deletion strains: AB43Δcrispr-cas, AB43Δcas3, AB43Δcsy1, AB43Δcsy3, AB43Δcsy4, showed significantly elevated of abaI mRNA (Fig. 6C). On the contrary, the abaI transcript level in each mutant complemented by the corresponding component were similar to those in AB43 (Fig. S5A). Then, we constructed abaI deletion mutant and tested its drug resistance. Compared to AB43, AB43ΔabaI was sensitive to all 23 tested drugs except piperacillin (Table 2). Moreover, qRT-PCR results showed that the mRNA expression of genes related to drug resistance, such as MFS (craA and rpoB), the RND superfamily (adeJ), TCS (pmrA, pmrB, adeS, bfmS, and bfmR) (45), biofilm formation (ompA and lpsB) (46), clinically significant cephalosporin resistance gene (bla_OXA-51-like) (47), and others (48 –51) in the AB43ΔabaI were reduced (Fig. 7). Beyond this, the biological traits associated with drug resistance are also altered (Fig. S6). Furthermore, double knockdown of crispr-cas and abaI was comparable to the abaI knockdown alone in antibiotic resistance profiles (Table 2, Fig. 7, and Fig. S6).

FIG 6 — AB43 crispr-cas deficiency increases the secretion of AHLs. (A) Schematic of predicted hybridization fragments between AbaI and CRISPR. A total of 11 bp of *abaI* mRNA hybridize with spacer 101. (B) AHL production in an entire CRISPR-Cas knockout mutant at different stages of growth in liquid LB medium. AHL in the culture supernatant of *A. baumannii* was quantitated using a β-galactosidase assay with *A. tumefaciens* KYC55 serving as the reporter strain. Atotal of 50 μg/mL of C₆HSL was taken as a positive control. (C) *AbaI* transcripts in AB43 and indicated *crispr-cas* deletion mutants were quantified by qRT-PCR. Based on three independent experiments, the results are expressed as the mean ± SEM (n = 3; one-way ANOVA with Tukey's *post hoc*; *, P < 0.05; ***, P < 0.001; NS, not significant).

FIG 7 — *AbaI* alters drug-resistant gene expression in AB43. (A–E) RNA was isolated when AB43, AB43Δ*abaI*, and AB43Δ*crispr-cas-abaI* grew to an OD₆₀₀ of 1.0 in liquid LB medium. Transcripts of indicated drug-resistant factors (A) ABC and MFS; (B) RND and SMR; (C) two-component system; (D) biofilm formation; and (E) other clinically relevant antibiotic-resistant genes in AB43-derived strains were quantified by qRT-PCR. This data represents the mean ± SEM from three independent experiments (two-way ANOVA with Tukey's *post hoc*; *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant).

We investigated the CRISPR-Cas system to understand which gene was necessary to regulate abaI. There were still eight A. baumannii with incomplete I-Fb CRISPR-Cas systems resistant to one or two category antibiotics that were tested in our collection of clinical isolates. We determined the cas gene expression of these eight strains and found that it compared with AB43; the cas gene expression level of these eight strains was significantly increased (Fig. S7). The initial results indicated that cas3, csy1, csy3, and csy4 were primarily involved in regulating abaI mRNA expression, and the AB43Δcas3 expression of abaI was uppermost among the single cas gene knockdown strains (Fig. 6C). Additionally, cas3-negative clinical strains had the highest resistance rates compared with cas3-positive in I-Fb strains (Table 1). Previous studies showed that Cas3 could target the QS regulator lasR mRNA to reduce bacterial virulence in P. aeruginosa (32). Hence, we deduce that abaI mRNA might be targeted by crRNA and cleaved by Cas3. From ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/), it is clear that AB43 does not have any acquired resistance genes. It only has the intrinsic ampC and bla_OXA-51-like genes. Consistent with our inference, ampC, and bla_OXA-51-like upregulated in all deletion strains except AB43Δcsy4, and the AB43Δcas3 expression of these two intrinsic drug-resistant genes was highest in all deletion strains (Fig. 8A and B). Similarly, qRT-PCR results showed that most of drug resistance genes: ABC transporters (macB, emrB) (34), MFS (craA, rpoB, tetB, abaQ), the RND superfamily (adeJ, adeB, adeG), the SMR protein family (abeS, abeM) (35), TCS (pmrA, pmrB, adeR, adeS, bfmS, bfmR, baeS) (45), biofilm formation (ompA, ompW, lpsB, abaI) (46), clinically significant cephalosporin resistance gene (ampC, bla_OXA-51-like) (47), and other genes (48 –51) in the AB43Δcrispr-cas and AB43Δcas3 were raised (Fig. 8C–J). We also noted that when one specific component of the CRISPR-Cas system was knocked out, the expression of the other components, including cas3, were also suppressed (Fig. S5). Taken together, these data indicated that the cleavage activity of Cas3 was the most critical factor in CRISPR-Cas system targeting abaI mRNA for degradation.

FIG 8 — *Cas3* is necessary for *abaI* mRNA repression in *A. baumannii*. (A and B) mRNA expression of *ampC* and *bla*_OXA-51-like in different deletion strains. RNA was isolated when AB43, a total CRISPR-Cas cluster deletion mutant, and single element deletion mutants, grew at an OD₆₀₀ of 1.0 in liquid LB medium, and transcripts for indicated genes were quantified by qRT-PCR. (C–E) mRNA expression of antibiotic-resistant genes related to (C) two-component systems; (D) biofilm formation; and (E) other clinically relevant antibiotic-resistant genes in AB43Δ*crispr-cas*. (F–J) mRNA expression of antibiotic-resistant genes (F) ABC and MFS; (G) RND and SMR; (H) two-component system; (I) biofilm formation; and (J) other clinically relevant antibiotic resistance genes in AB43Δ*cas3*. This data represents the mean ± SEM from three independent experiments (one-way ANOVA with Tukey's *post hoc*; *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant).

DISCUSSION

CRISPR-Cas system is a kind of bacterial immune system that enables bacteria to protect themselves against invasive mobile genetic elements which may carry the genes for antimicrobial resistance (AMR) (12). Many recent studies have shown that the CRISPR-Cas system is closely related to bacterial drug resistance (25, 52, 53). Our previous study observed cas gene expression changes in csy1 single knockout strains under antibiotic stress (27). However, how the CRISPR-Cas systems regulate antibiotic resistance in A. baumannii remains unknown. This study focused on a clinical strain AB43, using homologous recombination and sequentially knocking out each component and the entire CRISPR-Cas system to determine their effects on drug resistance. The most interesting finding was that deleting any component of the CRISPR-Cas system rendered AB43 significantly resistant to most of the tested drugs. Furthermore, we demonstrate that the I-Fb CRISPR-Cas system may target and degrade the abaI (QS synthase) mRNA, leading to drug resistance-related biological traits and genes being inhibited because of the low level of AHLs (Fig. 9). Moreover, Cas3 cleavage activity was the most critical factor in regulating abaI mRNA degradation.

FIG 9 — CRISPR-Cas in *Acinetobacter baumannii* contributes to antibiotic susceptibility by targeting endogenous *abaI* mRNA. Scheme summarizing the mechanisms in which CRISPR-Cas represses antibiotic resistance in *A. baumannii*. First, CRISPR-Cas targets *abaI* to increase ROS. An activated TCA cycle further accelerates the generation of ROS. Additionally, CRISPR-Cas targets *abaI* to suppress the efflux pump while also suppressing the energy required to drive it. CRISPR-Cas targeting *abaI* can also repress lipid A and biofilm formation. Finally, by targeting *abaI*, CRISPR-Cas also controls most multiple drug resistance genes.

The entire CRISPR-Cas represses antibiotic resistance in A. baumannii. Of the 245 A. baumannii clinical isolates, only two isolates were detected with a complete CRISPR-Cas system, and they were resistant to one category of antibiotics tested. All cas gene-negative strains had significantly higher resistance rates than positive strains. This inverse relationship between CRISPR-Cas and antibiotic resistance has been reported in Enterococcus faecalis and Klebsiella pneumoniae (54, 55). Nonetheless, there were still eight A. baumannii with incomplete I-Fb CRISPR-Cas systems resistant to one or two category antibiotics. Compared with AB43, the cas gene expression level of these eight strains was significantly increased. It is possible that when a certain Cas component of the I-Fb type is lost, other Cas may have an additional domain that can compensate for the role of a lost Cas (56, 57).

Additionally, most CRISPR-Cas positive strains have incomplete CRISPR-Cas systems. This may be because A. baumannii only selectively lost the cas gene cluster to adapt to environments with high concentrations of antibiotics, while the remaining incomplete CRISPR-Cas system could not give full play to its immune defense function. Other studies have shown that the CRISPR-Cas system can also be transferred among various bacterial genomes by inserting transposable elements (58), which can explain why some A. baumannii have incomplete CRISPR-Cas systems.

Cas3 is necessary for CRISPR-Cas targets abaI mRNA to repress antibiotic resistance. We found that with the AB43Δcas3 expression of abaI, two intrinsic genes ampC and bla_OXA-51-like were highest among the single cas gene knockdown strains. Additionally, cas3-negative clinical strains had higher resistance rates than cas3-positive in I-Fb strains. By analogy with that result, Cui et al.'s results suggest that Cas3 targets and downregulates the expression of the operon related to QS, resulting in the expression of Salmonella biofilm forming-related genes being changed (59). The Cas3 of P. aeruginosa recognizes and cleaves the mRNA for the bacterial QS regulator LasR to dampen the recognition of toll-like receptor 4 (TLR4) (32). Another interesting phenomenon was that when one specific component of the CRISPR-Cas system was knocked out, the expression of cas3 was suppressed. This further confirms our speculation that loss of either component will result in a decreased expression of cas3 and eventually lead to drug resistance. A similar phenomenon was found in 4,893 strains of A. baumannii collected in the NCBI (60).

CRISPR-Cas represses multidrug efflux pumps by targeting abaI. Our results showed that, in AB43Δcrispr-cas, EtBr efflux, efflux pump-related gene expression, and energy required for efflux pumps were significantly raised. While in AB43ΔabaI, EtBr efflux and energy required for efflux pumps was significantly reduced. Moreover, the expression of several efflux pump-related genes were also reduced in AB43ΔabaI and AB43Δcrispr-cas-abaI. The positive association between QS-controlled efflux pumps has been commonly identified in many bacterial pathogens. For instance, in Bacteroides fragilis, a self-inducing molecular receptor of the QS system reacts to exogenous AHL, upregulating the bmeB efflux pump's expression and developing antibiotic resistance (61). An autoinducer can upregulate the multidrug resistance pump MexAB-OprM, allowing bacteria to acquire multidrug resistance in P. aeruginosa (62).

At least two TCSs in A. baumannii have been reported to upregulate multidrug resistance efflux pumps. AdeRS activates the AdeABC multidrug resistance pump, while BaeSR activates AdeABC, AdeIJK, and MacAB-TolC (7, 63 –67). The qRT-PCR results demonstrated that in AB43ΔabaI and AB43Δcrispr-cas-abaI, adeR, baeS, and some efflux pump-related genes such as adeB are increased. The results were not as expected. Additionally, the expression of adeB is increased by about 5-fold in the AB43Δcrispr-cas mutant. The results of these two studies seem to be contradictory. This is probably because although AB43ΔabaI and AB43Δcrispr-cas-abaI were designed to be AHL-deficient, subsequent experiments with these mutants may have been influenced by the presence of abaI homologs; AbaI is similar to the LuxI family of autoinducer synthases (68). In addition, it is indicated that a combination of multiple genes may produce efflux pump phenotypes.

Biofilm formation and membrane permeability might also be a specific physiological aspect of CRISPR-Cas targeting abaI. AB43Δcrispr-cas formed a significantly more robust biofilm; the biofilm-related gene expression and lower membrane permeabilities were induced. In contrast, the biofilm and its related gene expression were significantly reduced in the AB43ΔabaI and AB43Δcrispr-cas-abaI strains. Biofilms can reduce the sensitivity to aminoglycosides (69), tetracycline, and macrolides by reducing the electrochemical gradient (70). Additionally, QS signals and the resulting downstream consequences can elicit physiological changes that alter the antimicrobial susceptibility of cells within a biofilm. Consistent with this, Niu et al. deleted the abaI in A. baumannii and found that biofilm formation was dramatically decreased in the mutant strain (71). In Streptococcus, competent stimulating factors can enhance the biofilm regulation by the QS system (72). As a regulator of Staphylococcus epidermidis biofilm formation, the QS system-related comprehensive regulator sarA plays an important role (73, 74).

Inhibition of the CRISPR-Cas system would affect AbaI-AHL complex formation, which might be responsible for a significant induction in catalase and SOD activities. This study found that AB43Δcrispr-cas had reduced generation of total ROS and increased SOD activity compared with AB43, while AB43ΔabaI had the opposite result. In A. baumannii ATCC 17978, inactivation of SOD decreased resistance to oxidative stress and susceptibility to antibiotics (75). QS also regulates the production of 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), a respiratory chain inhibitor that binds to the cytochrome bc₁ complex. This causes an accumulation of ROS, a decrease in membrane potential, and ultimately, autolysis. Autolysis of bacterial cells leads to the release and accumulation of extracellular DNA, enhancing biofilm formation and conferring resistance to positively charged antibiotics (76).

CRISPR-Cas also represses drug-resistant related genes by targeting abaI. β-lactamase is an effective resistance mechanism of A. baumannii that can inactivate β-lactam antibiotics (1). Based on the sequence homology, β-lactamases were classified into four types: class A extended-spectrum β-lactamases (ESBL), class B Metallo-β-lactamases (MBL), class C β-lactamases (AmpC) and Class D β-lactamases (OXA) (77). All four types of β-lactamases were reported in A. baumannii (78). Compared to AB43, various resistance genes were expressed at elevated levels in AB43Δcrispr-cas. It is striking that the intrinsic drug resistance gene ampC, often found in A. baumannii from China (79, 80), can be increased up to 200-fold. Another intrinsic drug resistance gene, bla_OXA-51-like was found to be elevated up to 80-fold. Nevertheless, the expression of ampC did not change and bla_OXA-51-like significant reduction in AB43ΔabaI and AB43Δcrispr-cas-abaI. A similar phenomenon has been previously reported. According to Dou et al., AHLs generated by A. baumannii might increase the expression of drug-resistance genes such as bla_OXA-51-like, ampC, adeA, and adeB (81).

This study revealed that CRISPR-Cas3 in A. baumannii contributes to antibiotic susceptibility by targeting endogenous genes abaI. Accordingly, this knowledge provides a new way to understand the functions of CRISPR-Cas systems in the drug sensitivity of A. baumannii. Our findings also provide vital information for the correlation study of CRISPR-Cas systems and A. baumannii drug resistance. However, the exact mechanism of how the CRISPR-Cas system targets abaI is not yet entirely distinct. It remains to be elucidated in future work whether CRISPR-Cas systems confer a similar susceptibility to antibiotics in other bacterial species as what has been observed in A. baumannii.

MATERIALS AND METHODS

Bacterial isolates.

A total of 245 clinical A. baumannii strains were randomly collected from five hospitals. These five sources and their isolates were as follows: 97 isolates from the Affiliated Hospital of Yangzhou University, harvested between 2017 to 2019; 84 isolates from the Affiliated Zhangjiagang Hospital of Soochow University, harvested between 2019 to 2020; 27 isolates from Northern Jiangsu People's Hospital, harvested between 2017 to 2018; 21 isolates from Xuyi People's Hospital, harvested between January and April 2019; and, 16 isolates from Wuxi People's Hospital, harvested between June and December 2018. The 245 clinical A. baumannii strains were isolated from sputum (n = 181, 73.81%), urine (n = 17, 7.14%), shunt fluids (n = 17, 7.14%), blood (n = 15, 5.95%), and other sources (n = 15, 5.96%). All bacterial strains were grown at 37°C on Luria-Bertani (LB) agar plates and subjected to identification by API20 NE (bioMérieux Marcy-l’Étoile, France) (82).

Detection of CRISPR-Cas systems.

PCR determined the prevalence of CRISPR-Cas systems. The DNA of all bacterial isolates were extracted by boiling using 1× TE solution (10 mmol/L Tris-HCl and 1 mmol/L EDTA; Sangon, China). PCR primers were designed to detect the CRISPR-Cas system using A. baumannii ATCC 19606 and AB43 as reference strains, listed in Table S1 (Supporting Information). PCR cycle was as follows: predenaturation at 95°C for 5 min, denaturation at 94°C for 1 min, annealing at the optimal temperature of different primers for 30 s, and extension at 72°C for 1 min. Denaturation, annealing, and extension steps were repeated for 35 cycles, with a final extension step at 72°C for 10 min. PCR products were separated and detected by gel electrophoresis and were confirmed by DNA sequencing (Sangon, China) and nucleotide BLAST search using GenBank.

Multilocus sequence typing (MLST).

MLST was performed according to a previously described study (83). Briefly, internal fragments of seven housekeeping genes (gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD) were amplified by PCR (84). Primers are listed in Table S2 (Supporting information). The sequences of these seven housekeeping genes were compared with existing sequences in the MLST database (http://pubmlst.org/abaumannii/) to assign allelic numbers. Sequence types (STs) were assigned according to their allelic profiles.

Antimicrobial susceptibility testing.

Based on Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2016), antibiotic resistance was assessed on Mueller-Hinton agar using the disk diffusion or broth microdilution method. All A. baumannii clinical isolates were tested for susceptibility to nine types of antimicrobial agents (penicillin, β-lactam/β-lactamase inhibitor combinations, cephems, carbapenems, aminoglycosides, fluoroquinolones, lipopeptides, tetracyclines, and folate pathway inhibitors). ATCC 19606 and ATCC17978 were used as controls. Isolates resistant to three or more classes of antibiotics were classified as multidrug-resistant.

Construction of AB43 deletion mutants and complemented strains.

As previously described, in-frame deletion mutants of CRISPR-Cas genes were made using a recombineering system for targeted genome editing (85). Briefly, using AB43 genomic DNA and PKD4 as templates, the upstream and downstream homology arms of the target fragment and the kanamycin cassette fragment with FRT site were amplified, respectively. Three PCR amplicons containing overlapping regions were assembled using overlap extension PCR with specific primers (Table S4), and the resulting fragment was electroporated into competent AB43 carrying pAT04, which expresses the RecAB recombinase. Transformants were selected on LB plates containing 7.5 μg/mL kanamycin, and PCR confirmed integration of the resistance marker. To remove the kanamycin resistance cassette, electrocompetent mutants were transformed with pAT03 plasmid, which expresses the FLP recombinase. A loss of kanamycin resistance was observed in these colonies, confirmed by PCR, and sequenced using identification primers (Table S4).

Complementation vectors for the deletion strains were constructed using the primer sets in Table S4. The full-length genes with their native promoters were amplified, cloned into vector PABBR-MCS (85), and electroporated into corresponding mutant strains.

In vitro growth kinetics sssays.

Overnight AB43 cultures of all strains were prepared and equalized by dilution adjustments to assess the growth of AB43 strains in LB. Optical density at 600 nm (OD₆₀₀) was used to measure the cell densities.

Crystal violet staining.

We measured the biofilm formation of A. baumannii by staining with crystal violet (86). Overnight cultures of A. baumannii strains were diluted 1:100 in fresh liquid LB and grown to OD₆₀₀ = 0.5 (5 × 10⁸ CFU/mL). Next, A. baumannii strains were cultured in sterile 96-well microtiter plates for 24 h at 37°C. After incubation, the supernatant planktonic bacteria were removed, and the wells were washed three times with phosphate-buffered saline (PBS). The biofilm was fixed with 4% paraformaldehyde and then washed again. The residual biofilms were then stained with 200 μL of 0.1% (1 g/L) crystal violet and incubated for 10 min with gentle agitation. The plates were washed twice with PBS, and 200 μL of 30% acetic acid was added to solubilize the dye. Finally, the acetic acid was transferred to a new plate, and the absorbance at 570 nm (BioTek, USA) was recorded (86). This experiment performed three technical replicates, and averaged the results. LB broth uninoculated with bacteria was used as a negative control.

Cell membrane permeability assessment.

Overnight cultures of A. baumannii strains were adjusted to an OD₆₀₀ of approximately 0.5. Next, 10 nM propidium iodide (PI) was added to all samples, and the suspensions were incubated at 37°C for 20 min. After washing three times with PBS, we used an excitation wavelength of 535 nm and an emission wavelength of 615 nm to measure the fluorescence intensity of 10 nM PI-labeled cells in A. baumannii strains (87).

Outer membrane permeability assessment.

The fluorescent probe N-Phenyl-1-naphthylamine (NPN) (10 μM) (88) was used to evaluate the outer membrane permeability of A. baumannii strains. Overnight cultures were harvested by centrifugation, washing, and resuspension in PBS to OD₆₀₀ = 0.5. The dye NPN was added to a final concentration of 10 μmol/L. Cells were then incubated at 37°C for 30 min, and the fluorescence was measured using a Tecan Infinite M200 Microplate Reader. The emission wavelength was 350 nm, and the excitation wavelength was 420 nm.

Efflux pump assays.

An EtBr efflux assay was performed based on a previous study to test the effect of CRISPR-Cas on the inhibition of efflux pumps (89). Strains were resuspended with PBS to an OD₆₀₀ of 0.5. Cells were then coincubated with EtBr (8 μg/mL final concentration) or the known efflux pump inhibitor CCCP (10⁻⁴ M) at 37°C. We then centrifuged the pellets at 5,000 g for 10 min at 4°C and resuspended these pellets in fresh liquid LB. We measured the EtBr efflux in cells for 60 min using a 530-nm excitation wavelength and a 600-nm emission wavelength.

Membrane depolarization assays.

3,3′-Dipropylthiadicarbocyanine iodide (DiSC3(5), 0.5 μM) was utilized to determine membrane potentials (90). A. baumannii bacterial cells were washed and resuspended to obtain an OD₆₀₀ of 0.5 with PBS. DiSC3(5) was added (Aladdin, catalogue number D131315; ≥98%, 0.5 μmol/L) to the mixture. The dissipated membrane potential of A. baumannii strains was measured with an excitation wavelength of 622 nm and an emission wavelength of 670 nm.

Proton motive force assessment.

The proton motive force of A. baumannii strains was measured with the pH-sensitive fluorescence probe BCECF-AM (2 × 10⁻⁵ M). After 30 min of incubation at 37°C, the fluorescence spectrometer's excitation and emission wavelengths were set to 500 and 522 nm, respectively, to monitor the proton motive force of A. baumannii strains (91).

ATP level determination.

Intracellular ATP levels of A. baumannii strains were determined using an Enhanced ATP assay kit (Beyotime, China). Overnight cultures of strains were resuspended to obtain an OD₆₀₀ of 0.5 with 0.01 mol/L PBS (pH 7.4). Then the bacterial precipitates were lysed by lysozyme and centrifuged to be used to measure intracellular ATP levels. A 96-well plate containing the detecting solution was incubated at room temperature for 5 min. Supernatants were added and mixed immediately, and an Infinite M200 Microplate reader (Tecan) was used to monitor luminescence signals (92).

Total ROS and H₂O₂ measurement.

As previously described (37), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM) was applied to monitor the levels of ROS in A. baumannii strains following the manufacturer's instructions (Beyotime, China). After incubation at 37°C for 30 min, 200 μL of probe-labeled bacterial cells was added to a 96-well plate, and fluorescence units were immediately measured with an excitation wavelength of 488 nm and emission wavelength of 525 nm using an Infinite M200 Microplate reader (Tecan). The production of H₂O₂ in A. baumannii strains was assessed using a Hydrogen Peroxide assay kit (Beyotime, China). The absorbance of lysis buffer at 560 nm was measured after 1 h of incubation.

SOD activity assessment.

Intracellular superoxide dismutase (SOD) activity in A. baumannii strains was measured using the Total Superoxide Dismutase assay kit with WST-8 (S0101, Beyotime, China). Based on the manufacturer's instructions, 450 nm was used to measure absorbance.

TCA cycle measurements.

The intracellular concentrations of NAD⁺ and NADH were determined using an enzymatic cycling assay kit (Beyotime, China). A. baumannii was collected and diluted to an OD₆₀₀ of 0.5 in PBS and then resuspended in 200 μL of precooled extraction buffer. The lysate was centrifuged at 12,000 g for 10 min at 4°C, and the absorbance of the supernatant at 450 nm was measured. The concentrations were measured in quadruplicate from three independent experiments.

RNA isolation and qRT-PCR.

RNA isolation, cDNA synthesis, and PCR amplification were carried out as described previously (93). A. baumannii were grown overnight in LB broth and diluted 1:100 into 5 mL fresh LB supplemented with indicated antibiotics. After bacterial cells were grown to an OD₆₀₀ of 1 at 37°C, bacterial cells were harvested for total RNA using the RNAprep pure bacteria kit (TIANGEN, Beijing, China), and total RNA was assessed using the ratio of 260 nm/280 nm absorbance on a Nanodrop spectrophotometer. The RNA extracted from all bacterial cells was adjusted to equal concentrations before cDNA synthesis. According to the manufacturer's protocol, the reverse transcription of 1 μg extracted RNA was performed using a PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Beijing, China), and 10 ng of cDNA was used as a template for qRT-PCR. All primers used in this study for qRT-PCR are listed in Table S12 (Supporting information). Three independent qRT-PCRs were performed using the 7500 Fast real-time PCR system (Applied Biosystems, CA, USA). SYBR green I fluorescence in every cycle was monitored by the system software, and the threshold cycle (CT) was determined. 16S rRNA was used as an internal control, and the relative gene expression level was calculated using the 2^−ΔΔCt method (94).

Transcriptomic analysis.

Strains were grown in liquid LB to the early exponential phase. We extracted total RNA from these samples using an EASY Spin Plus kit (Qiagen, Hilden, Germany). We quantified RNA based on the ratio of absorbance (260 nm/280 nm) using a Nanodrop spectrophotometer (Thermo Scientific) and sequenced samples by using the Illumina Hiseq system (Sangon, Shanghai, China). According to the manufacturer's protocol, the library construction of purified mRNA was conducted using an Illumina Truseq RNA sample prep kit. The raw read counts were normalized with DESeq2 (95) to estimate gene expression and identify differentially expressed genes. Differential gene expression was identified using a threshold of P < 0.05, a fold change of at least two, and a false discovery rate (FDR) < 0.2. Differences between two A. baumannii strains were analyzed using Cuffdiff software (http://cufflinks.cbcb.umd.edu/).

N-Acyl homoserine lactone (AHL) quantification by β-galactosidase assay.

AHL levels were quantified in cell-free culture media taken from AB43 strains at various times using a method described by previous work (96), which involves the use of the β-galactosidase reporter strain A. tumefaciens KYC55 (68). The culture supernatant was obtained by centrifugation, and it was extracted three times with equal amounts of acidified ethyl acetate (0.1% vol/vol glacial acetic acid). We dried the extracts in a fume hood, resuspended them in 1 mL of acidified ethyl acetate, and then dried them again in a fume hood. In order to rehydrate the dried extracts, 1 mL of acetonitrile was used. The positive control was 50 μL of synthesized N-hexanoyl-L-homoserine lactone (C6-HSL) at 50 μg/mL, and the negative control was blank LB media. All experiments were repeated at least three times.

Statistical analyses.

Statistical analysis was performed using GraphPad Prism 8 and SPSS software. All data are presented as the mean ± SEM. The endpoint differences for growth curves (OD₆₀₀) were analyzed using a two-tailed paired t test. Unless otherwise noted, an unpaired t test between two groups or one-way ANOVA between multiple groups was used to calculate P-values (*, P < 0.05; **, P< 0.01; ***, P < 0.001).

Data analysis.

The whole genome sequence of A. baumannii strain AB43 was submitted to GenBank under accession number CP083181. The transcriptome sequencing data described was submitted to the NCBI Sequence Read Archive under accession number SRR17299399.

Supplementary Material

Reviewer comments

reviewer-comments.pdf^{(475KB, pdf)}

ACKNOWLEDGMENTS

We thank Bryan W. Davies from University of Texas at Austin and Yajun Song from the Beijing Institute of Microbiology and Epidemiology Academy of Military Medical Sciences for providing plasmids to construct the deletion and complemented mutants. We thank Hui Wang (Nanjing Agricultural University, China) for the kind donation of the biosensor strain A. tumefaciens KYC55.

This work was supported by the National Natural Science Foundation of China (82073611, 82002186).

We declare that this research was conducted without any commercial or financial relationships construed as a potential conflict of interest.

Y.W. and G.L. designed the experiments; Y.W., J.Y., X.S. and M.L. conducted the experiments; Y.W., J.Y., X.S., P.Z., and Z.Z. analyzed the data, prepared the figures and tables, and drafted the manuscript; Y.W., T.G., H.J., and G.L. revised and approved the final manuscript.

Footnotes

Supplemental material is available online only.

Supplemental file 1

Supplemental material. Download spectrum.00829-22-s0001.pdf, PDF file, 2.1 MB^{(475KB, pdf)}

Contributor Information

Guocai Li, Email: gcli@yzu.edu.cn.

Monica Adriana Garcia-Solache, Brown University.

Jiaqi Cheng, Keck School of Medicine at USC.

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PERMALINK

CRISPR-Cas in Acinetobacter baumannii Contributes to Antibiotic Susceptibility by Targeting Endogenous AbaI

Yuhang Wang

Jie Yang

Xiaoli Sun

Mengying Li

Pengyu Zhang

Zhongtian Zhu

Hongmei Jiao

Tingting Guo

Guocai Li

Roles

ABSTRACT

INTRODUCTION

RESULTS

Detection of CRISPR-Cas systems in A. baumannii isolates.

FIG 1.

TABLE 1.

TABLE 2.

Complete CRISPR-Cas system represses antibiotic resistance in A. baumannii.

Transcriptomic analysis of the entire CRISPR-Cas knockout mutant.

FIG 2.

CRISPR-Cas inhibits A. baumannii efflux pumps.

FIG 3.

CRISPR-Cas deletion impacts A. baumannii biofilm formation and membrane permeability.

FIG 4.

CRISPR-Cas contributes to reactive oxygen species generation in A. baumannii.

FIG 5.

CRISPR-Cas represses drug resistance by targeting abaI mRNA.

FIG 6.

FIG 7.

FIG 8.

DISCUSSION

FIG 9.

MATERIALS AND METHODS

Bacterial isolates.

Detection of CRISPR-Cas systems.

Multilocus sequence typing (MLST).

Antimicrobial susceptibility testing.

Construction of AB43 deletion mutants and complemented strains.

In vitro growth kinetics sssays.

Crystal violet staining.

Cell membrane permeability assessment.

Outer membrane permeability assessment.

Efflux pump assays.

Membrane depolarization assays.

Proton motive force assessment.

ATP level determination.

Total ROS and H2O2 measurement.

SOD activity assessment.

TCA cycle measurements.

RNA isolation and qRT-PCR.

Transcriptomic analysis.

N-Acyl homoserine lactone (AHL) quantification by β-galactosidase assay.

Statistical analyses.

Data analysis.

Supplementary Material

ACKNOWLEDGMENTS

Footnotes

Contributor Information

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Total ROS and H₂O₂ measurement.