Cas3 of type I-Fa CRISPR-Cas system upregulates bacterial biofilm formation and virulence in Acinetobacter baumannii by affecting cilia-related and ompA genes expression

doi:10.21203/rs.3.rs-4658723/v1

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Cas3 of type I-Fa CRISPR-Cas system upregulates bacterial biofilm formation and virulence in Acinetobacter baumannii by affecting cilia-related and ompA genes expression

https://doi.org/10.21203/rs.3.rs-4658723/v1

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Acinetobacter baumannii (A.baumannii) is an important pathogen causing various nosocomial infections. CRISPR-Cas system is the adaptive immune system of bacteria, which is also closely related to the drug resistance and virulence of bacteria. In this study, we successfully constructed a cas3 deletion mutant (19606Δcas3) and complemented strain (19606Δcas3/pcas3) to study the regulatory mechanism of type I-Fa cas3 on bacterial virulence. Our results showed that deletion of cas3(type I-Fa) significantly reduced the biofilm formation, virulence and pathogenicity to mice, the organ bacterial load of mice infected with cas3 deletion strain was significantly reduced; the lung inflammation was slightly changed; and the serum cytokine level was also decreased. The results demonstrated that cas3 enhanced the virulence and pathogenicity of A.baumannii. Mechanism analysis shown that deletion of cas3 can lead to the down-regulation of virulence factors such as biofilm formation related factors and outer membrane protein A(ompA). In addition, cas3 was also involved in the regulation of carbon metabolism and oxidative phosphorylation pathway of A.baumannii.

Acinetobacter baumannii

virulence

cas3

CRISPR-Cas system

mechanism

Acinetobacter baumannii is an important gram-negative non-fermenting opportunistic pathogen that causes hospital-acquired pneumonia and ventilator-associated pneumonia easily in hospital patients with low immunity[1]. Due to the rapid development of its antibiotic resistance, the World Health Organization has listed carbapenem-resistant A.baumannii as one of the pathogens in urgent need of exploring new drugs[2]. In addition to studying its drug resistance mechanism, more and more virulence-related studies on A.baumannii have been conducted in recent years. The major virulence factors found include adherence(Ata, TFP)[3, 4], effector delivery system(T2SS,T6SS)[5, 6], exotoxin(phospholipase C, phospholipase D)[7, 8], exoenzyme(CpaA)[9], immune modulation(capsule, LPS, ompA, pbpG)[10–13],biofilm(adeFGH efflux pump, Bap, Csu fimbriae, PNAG, Quorom sensing)[14–18], nutritional/metabolic factor(Acinetobactin, HemO cluster)[19, 20]. With the absence of some virulence factors, the pathogenicity of the highly virulent strain in the mouse infection model is remarkably reduced[21]. Therefore, the regulation of different virulence factors may point to a new direction in the treatment of A.baumannii infection.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is an acquired immune system widely present in genomes of archaea and bacteria, which acts as a defense mechanism against the invasion of exogenous nucleic acids such as phages and plasmids[22]. CRISPR-Cas systems consists of the CRISPR arrays, leader sequences, and CRISPR-associated (cas) genes. Type I CRISPR-Cas system is the most widely distributed in bacteria, and Type I-F CRISPR-Cas system is the main type existing in A.baumannii[23]. Type I-F CRISPR-Cas system is characterized by the fusion of Cas2 and Cas3 (Cas2/3), as well as the integration of the Cas1-mediated spacer to the CRISPR site[24]. The cas gene of the I-Fa type CRISPR-Cas system contains cas1, cas3, csy2, csy3 and csy6(Fig. 1). Cas3 is the marker of type I CRISPR-Cas system, which encodes a protein that may form cascade-like complexes with different compositions, in addition, it encodes a large protein with independent helicase and deoxyribonuclease activities [25].

Apart from its role in adaptive immunity, CRISPR-Cas system also plays an important role in regulating gene expression, especially in the regulation of bacterial virulence and population behavior[26]. Previous studies have shown that the CRISPR-Cas system can regulate the synthesis of outer membrane proteins of Salmonella typhi by regulating the outer membrane protein synthesis gene OmpR[27]. The biofilm formation, virulence and pathogenicity of Salmonella are reduced after cas3 deleted in Salmonella [28]. Jose Solbiati et al. found that virulence of the pathogen Porphyromonas gingivalis is controlled by the CRISPR-Cas protein Cas3[29]. These all indicated that CRISPR-Cas system might be a potential virulence regulatory factor. Our previous research mainly focused on the effect of type I-Fb CRISPR-Cas system on the biological traits in A.baumannii[30, 31], and conducted the whole genome analysis of the related strains[32]. We found that different subtypes (I-Fa and I-Fb) cas gene had low homology and identity, the results showed that complete I-Fb CRISPR-Cas system could inhibit the drug resistance and reduce the virulence of bacteria[31]. However, the role of type I-Fa CRISPR-Cas system in the drug resistance and virulence of A.baumannii is still unclear. Cas3 is a key protein of I-F CRISPR-Cas system, in order to further understand the role of cas3 gene in the virulence of A.baumannii, we constructed type I-Fa cas3 gene knockout and complementary strains in A.baumannii ATCC19606, explored the effect of cas3 gene on bacteria growth, biofilm formation, adhesion and invasion. We also established a bacteremia model of A.baumannii infection in mice to evaluate the role of cas3 in A.baumannii infection. In conclusion, our results indicated that type I-Fa cas3 is essential for the biofilm formation and virulence of A.baumannii.

2.1Deletion of cas3 gene does not affect growth or intracellular survival

We successfully constructed the 19606Δcas3 mutant and 19606Δcas3/pcas3 complement strains, which were verified by PCR and sequencing (Figure S1, S2). The ATCC19606, 19606Δcas3 and 19606Δcas3/pcas3 strains were cultured in a shaking table, and the standard growth curves were measured for 24 h. According to the OD₆₀₀ value and the number of viable bacteria, the growth of ATCC19606, 19606Δcas3 and19606Δcas3/pcas3 shown no significant difference (Fig. 2).

2.2Deletion of cas3 gene reduces bacterial biofilm formation

Bacterial biofilm is an important part of bacteria against external pressure[33]. In order to evaluate the effect of cas3 gene on bacterial biofilm formation, the biofilm formation ability was quantified by crystal violet staining, and the results showed that both ATCC19606 wild strain and the 19606Δcas3 mutant strain could form biofilm. However, the ability to form biofilms after cas3 gene knockout was significantly reduced, and the complementary strain restored the ability (Fig. 3A and B). As shown in Fig. 3C, the fluorescence intensities of ATCC19606 and 19606Δcas3/pcas3 strains were similar under confocal laser scanning microscopy. In addition, the fluorescence intensity of 19606Δcas3 strain biofilm was lower. Therefore, cas3 gene of type I-Fa CRISPR-Cas system is an important factor affecting the biofilm formation of A. baumannii.

2.3Deletion of cas3 gene decreases virulence and pathogenicity of A. baumannii

As the knockout of cas3 gene resulted in a decrease in the biofilm formation ability of the strain, and biofilm is also a virulence-related factor that can avoid immune-mediated clearance and establish persistent infection, thus we speculated that the virulence of 19606Δcas3 may also be changed. The virulence of bacteria is closely related to the adhesion and invasion of bacteria to cells. A549 cells were infected with strains of 19606WT, 19606Δcas3 and 19606Δcas3/pcas3 with MOI of 100, respectively, and their adhesion and invasion ability to mammalian cells were observed. As shown in Fig. 4, compared with 19606WT and 19606Δcas3/pcas3, the adhesion rate and invasion rate of strain 19606Δcas3 were significantly reduced.

To evaluate the effect of cas3 gene on the virulence of A.baumannii, an infection model of Galleria mellonella was established and the survival was observed. The same amount of bacteria (1.0×10⁶ CFU) were injected into each group. Results were shown in Fig. 5A, as we can see that a total of 90% larvae infected with ATCC19606 strain died within 12 hours, while only 20% of the larvae infected with 19606Δcas3 strain died. After 24 hours, all larvaes infected with ATCC19606 strain and the complementary strain were died, while those infected with 19606Δcas3 strain maintained a 50% survival rate after 96 hours.

Next, the mice were intraperitoneally injected with the bacterial suspension to construct a systemic infection model and further understand the virulence changes. After 24 h infection, we observed that the bacteria load of each organs in infected 19606Δcas3 mice was significantly lower than that in infected ATCC19606 and 19606Δcas3/pcas3 strains (Fig. 5B). The changes of pneumonic inflammation and injury were observed by hematoxylin and eosin (H&E) staining. As shown in Fig. 6B, the alveolar structure in the lung of ATCC19606 infected mice was filled with neutrophil infiltration and edema, and the alveolar structure was incomplete. However, the injury was milder in mice infected with 19606Δcas3 and was not observed in non-infected mice. The H&E staining results in the 19606Δcas3/pcas3 strain infection group were consistent with the observation and analysis results in the ATCC19606 infection group. In addition, enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of cytokines (interleukin − 1β [IL-1β], IL-6, and tumor necrosis factor-α [TNF-α]) in mice serum. Results showed that IL-1β, IL-6, and TNF-α in the 19606Δcas3-infected mice were reduced significantly compared to the ATCC 19606 or 19606Δcas3/pcas3-infected mice (Fig. 6A). In conclusion, our results demonstrate that the presence of cas3 gene could enhance the virulence and pathogenicity of A.baumannii.

2.4RNA-seq to explore the potential regulatory mechanism of cas3

In order to further understand the mechanism of cas3 gene in regulating biofilm and virulence, we performed transcriptome sequencing analysis. After cas3 gene knockout, other related cas genes in CRISPR-Cas system genes were up-regulated except for cas3 and cas6 genes, as shown in Table 1. Compared with the ATCC19606 strain, 539 genes in 19606Δcas3 strain were differentially expressed, including 272 genes up-regulated and 267 genes down-regulated (Fig. 7A), suggesting that cas3 gene were associated with multiple vital activities of bacteria. The differential expressed genes (DEGs) between ATCC19606 and 19606Δcas3 strains were evaluated according to Gene Ontology (GO) classification to investigate potential biological functions in which DEGs might be involved (Fig. 7C), it mainly showed the enrichment of differentially expressed genes in biological process, cellular component and molecular function on GO term. In the classification of biological processes, differential genes are mainly enriched in redox, fatty acid metabolism, cation transport, monocarboxylic acid transport, metal ion transport and other processes. In the classification of cell composition, it is mainly enriched in protein complex, cell membrane, mitochondrial membrane and other components. In molecular function, it is mainly enriched in oxidoreductase activity, adenine flavin dinucleotide binding, 3-hydroxyacyl-CoA dehydrogenase activity, ion transmembrane transporter activity and other functions.

Different genes usually coordinate with each other to exercise their biological functions in microbiology, through the significant pathway enrichment in Kyoto Encyclopedia of Genes and Genomes (KEGG) database, we can identify the most important biochemical metabolic pathways and signal transduction pathways involved in the differentially expressed genes. As shown in Fig. 7D, the main pathway changed was microbial metabolism in diverse environments, carbon metabolism, pyruvate metabolism and benzoate degradation, and the top 20 pathways are shown in the figure. We also found that genes related to oxidative phosphorylation pathway are significantly down-regulated, and oxidative phosphorylation is the pathway of ATP synthesis, so we detected the intracellular ATP of ATCC19606, 19606Δcas3 and 19606Δcas3/pcas3. The result is consistent with our conjecture, intracellular ATP content of 19606Δcas3 decreased significantly (Figure S3).

Our results showed that the deletion of cas3 gene had a strong impact on the biofilm and virulence. Analysis of transcriptome results also indicated that the expression of biofilm and virulence genes were down-regulated after cas3 gene knockout, as shown in Table 2. The gene expression levels of bacterial cilia-related proteins of csuA, csuB, csuC and csuE significantly decreased, and the cilia protein closely related to the biofilm formation and virulence. Notably, the expression levels of OmpA family proteins and two-component regulatory system BfmSR were also down-regulated, and the results were verified by qRT-PCR (Fig. 8). Therefore, RNA-seq further indicated that cas3 gene could regulate the bacterial virulence and biofilm formation through affecting the expression of related genes.

Table 1

Cas genes expression of ATCC19606Δ*cas*3
Locus-tag	Gene	Protein	log2 Fold Change(Δcas3/WT)	P-value
H0N29_14350	cas3	type I-F CRISPR-associated helicase Cas3	-8.0022	3.212E-27
H0N29_14380	cas1	type I-F CRISPR-associated endonuclease Cas1	0.57393	0.0029863
H0N29_14365	csy2	type I-F CRISPR-associated protein Csy2	1.1589	4.37E-20
H0N29_14370	csy3	type I-F CRISPR-associated protein Csy3	0.41792	3.13E-06
H0N29_14375	cas6/csy4	type I-F CRISPR-associated endoribonuclease Cas6/Csy4	-0.19228	0.89166

Table 2

Expression of virulence genes in the 19606∆*cas3* strain versus ATCC19606 strain.
Locus-tag	Gene	log2 Fold Change	P-value	Protein and function
H0N29_11295	OmpA	-1.8528	0.091911	OmpA family protein
H0N29_15280	bfmR	-0.83203	4.99E-14	response regulator transcription factor BfmR
H0N29_15275	bfmS	-0.66782	2.31E-07	sensor histidine kinase BfmS
H0N29_06290	csuE	-4.9714	1.73E-98	Csu fimbrial tip adhesin CsuE
H0N29_06270	csuA	-3.7801	2.18E-25	Csu fimbrial biogenesis protein CsuA
H0N29_06275	csuB	-3.7334	5.57E-58	Csu fimbrial biogenesis protein CsuB
H0N29_06280	csuC	-4.9373	1.13E-219	Csu fimbrial biogenesis chaperone CsuC

In recent years, multi-drug resistant rates of A.baumannii has gradually increased, and MDR bacteria can tolerate a variety of adverse stimuli to adapt to the environment and survive easily[34]. CRISPR-Cas system plays an important role in the adaptive immune activity of bacteria, its main function is defensing against bacteriophage, plasmids and other foreign DNA[22]. Recent studies have shown that CRISPR-Cas system not only protects the host against mobile genetic elements, but also regulates other physiological functions of bacteria, such as virulence, biofilm formation, drug resistance and quorum sensing[26]. Jianjian Jiao et al. found that Cas proteins were secreted virulence factor which can activate the host immune response in Mycobacterium tuberculosis[35]. Our research discovered the unconventional function of A.baumannii type I-Fa CRISPR-Cas system, cas3 enhanced the biofilm formation ability of the strain by regulating OmpA family proteins and Csu-related genes, making it easier for the strain to adhere and invade the host, thus enhancing its own virulence.

In this study, we studied the effect of cas3 gene on the biological activity of A. baumannii by knocking out the cas3 gene. Results first showed that the deletion of cas3 gene did not affect its growth, indicating that cas3 gene was not a necessary gene for bacterial growth. Biofilm is one of the population behaviors of bacteria, which helps bacteria stand up to external environmental pressure[36, 37]. Through the biofilm formation assay, the biofilm formation ability of 19606Δcas3 strain was decreased compared with ATCC19606. Similar with a previous study showed that biofilm formation of the Δcas3 Streptococcus strain (type I-C CRISPR-Cas system) was reduced[38]. Also, type I-E CRISPR-Cas Salmonella typhimurium wild strain has a stronger biofilm formation ability compared with the Δcas3 strain [28].

The effect of cas3 gene on the virulence of A. baumannii was explored through the infection model of Galleria mellonella and mice. Results showed that the virulence and pathogenicity of A. baumannii decreased after cas3 gene knocked out. Previous studies have also shown that CRISPR-Cas can enhance or reduce the virulence of bacteria. For example, cas9 could enhance the virulence by inhibiting its transcription regulatory factor regR in Streptococcus agalactiae [39]. Type I-F CRISPR-Cas system of Pseudomonas aeruginosa PA14 can change its virulence by targeting and inhibiting LasR, and cas3 gene knockout increases the toxicity of PA14 and the host's innate immune response[40].

After the deletion of cas3 gene, the expression of many genes was up-regulated or down-regulated, indicating that cas3 might be involved in the regulation of other bacterial life activities. Cas3 has an important influence on the expression of several Outer membrane protein A (OmpA) family protein. OmpA is a major component of outer membrane proteins (OMPs) in gram-negative bacteria, which is a key virulence factor. Overexpression of OmpA was strongly associated with severe hospital infection, bacteremia, and pneumonia infection[41]. Similarly, Medina-Aparicio L et.al. studied the CRISPR-Cas system in Salmonella spp. and showed that the CRISPR-Cas system could participate in the regulation of OmpR gene synthesis of Salmonella typhi[27]. It suggested us that regulation of bacterial virulence by cas3 could be target to OmpA family protein. We also found that the largest number of gene changes was in oxidation reduction process in GO terms (Fig. 7C), KEGG analysis showed that oxidative phosphorylation pathway was inhibited after cas3 gene deletion. The bacterial oxidative phosphorylation (OxPhos) system plays an essential role in energy production in the form of ATP, which has been considered to be a potential antibacterial drug target[42]. KEGG analysis showed that the oxidative phosphorylation pathway of A.baumannii was inhibited, and ATP production was decreased after cas3 gene deletion, which may be related to the weakening of virulence of 19606Δcas3.

In our previous study, we found that the resistance, biofilm formation ability, and virulence of the strain were up-regulated after any cas gene knocked out in the type I-Fb CRISPR-Cas system[43]. However, the opposite situation appeared after the cas3 gene knocked out in the type I-Fa CRISPR-Cas system in this study, which indicated that two subtypes of CRISPR-Cas system in A. baumannii played different regulatory roles in resistance and virulence. It is still necessary to continue to study how the CRISPR-Cas system regulates these functions. Exceedingly, all these evidences indicated that cas3 gene and CRISPR-cas system are closely linked to the virulence of A. baumannii, which maybe a therapeutic target in the future.

4.1Bacterial strains, plasmids, and growth conditions

Bacterial strains, plasmids and primers used in this study are listed in Supplementary Table S1. A.baumannii and E.coli strains were grown in lysogeny broth (LB) at 37°C. To select colonies during the construction of mutant and complementary strains, antimicrobial agents, tetracycline (50 µg/ml), carbenicillin (75 µg/ml), kanamycin (50 µg/ml), were added to the growth medium.

4.2Construction of cas3 gene deletion strain ATCC19606∆cas3

The Δcas3 mutant strain was constructed by a marker-free gene deletion method using Acinetobacter baumannii ATCC19606 as previously described[30]. First, the kanamycin resistance gene fragment with FRT locus was amplified using pKD4 as the template, upstream and downstream fragments of cas3 were amplified using ATCC19606 as the template. The polymerase chain reaction (PCR) amplification products were identified by agarose gel electrophoresis and purified using Fast Pure gel DNA Extraction Mini Kit (VaZyme). Products of three stages were fused by fusion PCR, and the recombinant product was recovered by gel cutting and sequencing by Beijing Tsingke Biotech Co., Ltd. Then electrotransform the sequenced correct 1 µg fusion PCR product into ATCC19606-pAT04(plasmid pAT04 with flippase recombinase) competent cells (100µL) in a 2mm cuvette at 1.8 kV, transfer it to 4 mL liquid LB containing 2 mM IPTG culture for 4 hours, centrifuge at 4000 rpm for 10 minutes to collect bacteria. Incubate bacteria overnight on LB agar medium containing 50 g/mL kanamycin at 37℃. Positive clones confirmed insertion of the kanamycin resistance gene by screening primers.

The kanamycin resistance gene in mutant ATCC19606∆cas3::kan was then lost by the following procedure. First, the pAT03 (pMMB67EH with flippase recombinase) plasmid was electro transformed into the ATCC19606∆cas3::kan mutant. Then, the bacteria were incubated with liquid LB containing 2 mM IPTG for 1 hour. Positive clones were selected on LB agar plates containing carbenicillin (75 µg/mL) and incubated overnight at 37℃. cas3 gene knockout mutant was confirmed by PCR and sequencing.

4.3Construction of cas3 gene complementary strain ATCC19606∆cas3/pcas3

Complementation of cas3 gene in the gene knockout mutant was performed through constructing a recombinant plasmid by the pMMB67EH vector. A 3236 bp fragment of cas3 was amplified from ATCC19606 genome DNA and ligated to the pMMB67EH vector, the recombinant plasmid was electro transformed into ATCC19606∆cas3 competent cells. The ∆cas3 mutant transformed with the complementary vector was selected on LB agar plates containing 50 µg/mL kanamycin and 50 µg/mL tetracycline. Positive clones were verified by PCR and sequencing.

4.4Determination of growth curve

ATCC19606, ATCC19606∆cas3 and ATCC19606∆cas3/pcas3 cultured overnight were inoculated into fresh liquid LB broth at a ratio of 1: 1000, and cultured by shaking at 37℃. The optical density at 600 nm (OD₆₀₀) was measured every 2 hours after inoculation, bacteria counts were performed on LB plates after fold dilution in 96-well plates. Growth curve was drawn according to the results.

4.5Biofilm formation assay

The bacteria were cultured in a shaking table at 37℃ until the OD₆₀₀ value was 0.5 (the bacterial count was 1.0×10⁸ CFU/mL), inoculated in 200 µL to 96-well plates at a ratio of 1:100, quietly placed in an incubator at 37℃ for 48 hours. Then the bacterial solution was washed three times with PBS. After the 96-well plate was dried, it was dyed with 4% crystal violet (Solaribo) for 30 min in dark. Discard the staining solution and wash three times with PBS. Finally, the crystal violet dye adsorbed on the biofilm was dissolved with 95% ethanol, and the absorbance at 590 nm was measured. In this experiment, liquid LB medium without bacteria was used as the negative control.

4.6Confocal scanning laser microscopy (CSLM)

The bacteria were cultured until the OD₆₀₀ value was 0.5, and 1ml bacterial liquid was inoculated into a 24-well plate. Round coverslip was placed at the bottom of the hole and placed in an incubator at 37℃ for 48 h. Discard the bacterial liquid and rinse it twice with physiological salt solution. According to the manufacturer's instructions, the biofilm was dyed with BacLight Live/Dead Dyeing Kit (Invitrogen) for 15 minutes in the dark, observed under a laser confocal scanning microscope and analyzed by ZEN software.

4.7 Adhesion and invasion

The A549 cells were cultured in a 24-well cell culture plate to a cell density of about 3.0 × 10⁵ cells/well. A.baumannii strains were infected with a bacterium: cell ratio of infection multiple (MOI) of 100:1. The adhesion and invasion of strains to A549 cells were measured by colony-forming unit (CFU) counts. After the bacteria strains and cells were co-cultured for 2 h, the extracellular free bacteria were removed by washing with PBS three times, and treated with freshly prepared sterile 0.1% Triton X-100 for 15 minutes. The cell culture medium containing 32 g/mL tigecycline was replaced in the remaining wells and incubated for a further 90 min at 37℃ to kill bacteria adhering to the outside of the cells. Then discard the cell culture medium and wash the cells three times with PBS. Finally, 0.1% Triton X-100 was added to each well for incubation 10 minutes to release intracellular bacteria, and the bacterial CFUs assay was performed on agar plates.

4.8Infection model of Galleria mellonella

All the Galleria mellonella larvae used in the experiment were purchased from organisms of family origin in Yunnan Province. We randomly selected four groups of healthy larvae (10/group) with no evidence of blacking, weighing between 200 ~ 300 mg for subsequent infection. The Galleria mellonella were infected by right hind foot injection of 10 µl (about 1.0×10⁶ CFU) each, control group were injected with equivalent PBS. The number of dead larvae was observed and recorded every 12 hours.

4.9Mouse bacteremia model and cytokines determination

Eight-week-old female C57 mice were raised under conventional conditions. Cyclophosphamide was used to induce neutropenia in mice, as described previously(32). The neutropenic mice were infected with 5×10⁸ colony-forming units (CFU) of A.baumannii. The mice were sacrificed 24 hours after infection. Blood from the eyeball and organs were taken. The left lung and other organs were used for bacterial load count on LB agar plates. Right lung tissues were fixed in formalin solution and stained with hematoxylin and eosin (HE).

Cytokine in the serum of infected mice were detect by enzyme-linked immunosorbent assay (ELISA). Blood was taken from eyeball of each aforementioned mice. After blood was naturally coagulated at room temperature for 30 minutes, it was centrifuged at 1000 g for 10 min at 4 ℃. The supernatant(serum) was aliquoted into EP tubes and stored at − 20 ℃ for future use. ELISA were completed by Solarbio's kit.

4.10 RNA transcriptome sequencing and qRT-PCR

The bacteria RNA samples were subjected to quality detection, cDNA synthesis, cDNA fragment modification and sorting, library amplification, and the constructed cDNA library was sequenced by Illumina HiSeqTM. Technical services for this process and subsequent bioinformatics analysis were provided by Shanghai Shenggong Biotechnology Limited liability Company.

Bacteria used in qRT-PCR were cultured overnight to 10⁹ CFU/ml, 1 ~ 3 ml of bacterial culture was centrifuged, and the total RNA of bacteria was extracted by RNAprep Pure Bacteria Kit (TIANGEN, Beijing) according to the instructions. cDNA was obtained by reverse transcription of RNA with reverse transcription kit (Vazyme, Nanjing, China), which was packaged and stored at -20 ℃ for future use, and qRT-PCR was performed with ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China). Primers used are listed in Supplementary Table 1.

4.11 Statistical analysis

Data were presented as the mean standard deviation (SD) of the triplicate replicates. Mean comparisons between groups were performed using the two-tailed Student's t test. All statistical analyses were performed using GraphPad Prism version 8.4.2 (GraphPad Software Inc, San Diego, CA, usa), and a significant P value of < 0.05 was considered significant.

Competing interests: The authors declare that they have no competing interests.

Funding：This work was supported by the National Natural Science Foundation of China (82073611, 82373637, 82002186), the Jiangsu Provincial Natural Science Foundation of China (BK20231241), and the Open Project Program of Jiangsu Key Laboratory of Zoonosis (Grant number: R2201).

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Cas3 of type I-Fa CRISPR-Cas system upregulates bacterial biofilm formation and virulence in Acinetobacter baumannii by affecting cilia-related and ompA genes expression

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Abstract

Figures

1. Introduction

2. Results

2.1Deletion of cas3 gene does not affect growth or intracellular survival

2.2Deletion of cas3 gene reduces bacterial biofilm formation

2.3Deletion of cas3 gene decreases virulence and pathogenicity of A. baumannii

2.4RNA-seq to explore the potential regulatory mechanism of cas3

3. Discussion

4. Methods

4.1Bacterial strains, plasmids, and growth conditions

4.2Construction of cas3 gene deletion strain ATCC19606∆cas3

4.3Construction of cas3 gene complementary strain ATCC19606∆cas3/pcas3

4.4Determination of growth curve

4.5Biofilm formation assay

4.6Confocal scanning laser microscopy (CSLM)

4.7 Adhesion and invasion

4.8Infection model of Galleria mellonella

4.9Mouse bacteremia model and cytokines determination

4.10 RNA transcriptome sequencing and qRT-PCR

4.11 Statistical analysis

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References

Additional Declarations

Supplementary Files

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