The gigA/gigB Genes Regulate the Growth, Stress Response, and Virulence of Acinetobacter baumannii ATCC 17978 Strain

Acinetobacter baumannii is an important pathogen of nosocomial infection. Recently, a group of genes, named “gig” (for Growth in Galleria), have been identified in a contemporary multi-drug resistant clinical isolate of A. baumannii—strain AB5075. Among these so-called gig genes, gigA and gigB were found to promote antibiotic resistance, stress survival, and virulence of AB5075 by interacting with the nitrogen phosphotransferase system (PTSNtr). This study aimed to investigate the roles of gigA/gigB, which appear to comprise a stress-signaling pathway (encoding for an atypical two-component system response regulator and a predicted anti-anti-sigma factor, respectively), and the involvement of ptsP (encoding the Enzyme I component of the PTSNtr) in the growth, stress resistance, and virulence of the widely studied A. baumannii strain ATCC 17978. Genetic analyses of strains harboring mutations of gigA and gigB were performed to investigate the roles of these genes in bacterial growth, stress resistance, evading macrophage defense, and killing of Galleria mellonella larva. In contrast with findings from strain AB5075 where gigA and gigB contribute to aminoglycoside resistance, the data presented herein indicate that the loss of gigA/gigB does not impact antibiotic resistance of strain ATCC 17978. Interestingly, however, we found that deletion of gigA/gigB in the ATCC 17978 background imparts a general growth in laboratory medium and also conferred growth and replication defects within murine macrophages and an inability to kill G. mellonella larvae. Importantly, studies as well as the loss of ptsP restored the phenotypes of the gigA/gigB mutant to that of the wild-type. The data presented herein indicate that in A. baumannii ATCC 17978, the gigA/gigB genes play a key role in both growth and virulence traits, but are dispensable for other stress-resistance survival phenotypes, including aminoglycoside resistance. Our findings thus highlight several similarities and also important differences between the gigA/gigB stress-signaling pathway in two commonly studied isolates of this troublesome pathogen.


INTRODUCTION
Acinetobacter baumannii is a Gram-negative bacterium responsible for approximately 20% of intensive care unit infections worldwide and is the top-ranking pathogen on the World Health Organization's list of priority antibioticresistant pathogens (Lee et al., 2017;WHO, 2017;Karalewitz and Miller, 2018). Many circulating A. baumannii strains exhibit a multidrug-resistant phenotype due to a combination of intrinsic and acquired traits (Peleg et al., 2012;Gottig et al., 2014). Identification of virulence determinants and understanding of the mechanisms underlying the pathogenesis of A. baumannii are important for combating A. baumannii infection.
Recently, Gebhardt et al. (2015) have identified a group of genes, named "gig" (for Growth in Galleria), that are required for growth of the highly virulent and highly antibiotic resistant A. baumannii strain AB5075 in Galleria mellonella larvae. Among these genes, gigA and gigB were found to promote antibiotic resistance, stress survival, and virulence of AB5075 by interacting with the nitrogen phosphotransferase system (PTS Ntr ) (Gebhardt and Shuman, 2017). gigA encodes a protein phosphatase 2C-type phosphatase, and gigB encodes a putative anti-anti-sigma factor. GigA was shown to dephosphorylate GigB, which in turn regulates the phosphate level on NPr, a key component of the PTS Ntr . Disruption of the GigA/GigB signaling pathway led to the altered expression of numerous stress response genes. Thus, the intersection of GigA/GigB with the PTS Ntr promotes stress survival (Gebhardt and Shuman, 2017).
The ptsP gene encodes the enzyme I component of the PTS Ntr . Mutations in ptsP increases tobramycin resistance (Schurek et al., 2008;Scribner et al., 2020;Abisado et al., 2021). In AB5075, deletion of ptsP in either a gigA or gigB background suppresses the gig mutant phenotypes to near-wildtype levels, including restoration of aminoglycoside resistance, stress survival, and growth in Galleria larvae (Gebhardt and Shuman, 2017). Our previous work has revealed that in A. baumannii AB5075 mutants lacking both gigA and gigB (i.e., a gigAB double mutant), only concurrent complementation of both gigA and gigB can restore kanamycin resistance to wild-type levels, suggesting that gigA and gigB are inseparable in the pathogenesis of A. baumannii (Gebhardt and Shuman, 2017). However, the role played by ptsP in the survival and virulence of an A. baumannii gigAB mutant strain remains unknown.
ATCC 17978 is among the best-studied strains of A. baumannii and is an ideal model for genetic manipulation compared with clinical isolates due to its sensitivity to most antibiotics and high genome homology to current A. baumannii isolates (Sahl et al., 2011;Jacobs et al., 2014a). In this study, we investigated the roles of gigA/gigB and the involvement of ptsP in the growth, stress response, and virulence of ATCC 17978. Our results may provide new information about the roles of gigA/gigB and the PTS Ntr system in the pathogenesis of A. baumannii infection.

Bacterial Strains and Culture
A. baumannii ATCC 17978 was purchased from The American Type Culture Collection (Manassas, VA, United States). Escherichia coli DH5α was obtained from Invitrogen (Carlsbad, CA, United States). The tetracycline-resistant and sucrosesensitive plasmid pMJG42, apramycin-resistant pMJG120, and gentamicin-resistant pMJG125 plasmids were kept in our laboratory at the University of Chicago (Chicago, IL, United States). The bacteria were cultured in lysogeny broth (LB) medium at 37 • C. When required, the antibiotics added for selection were tetracycline (10 µg/mL), apramycin (50 µg/mL), and gentamicin (10 µ g/mL).

Generation of Gene Deletion and Complementation Plasmids
Gene deletion and complementation plasmids were generated as previously described (Jacobs et al., 2014b;Gebhardt et al., 2015;Gebhardt and Shuman, 2017). Briefly, gene deletions were performed using allelic exchange plasmid pMJG42. The resulting plasmids (pMJG42-gigAB, pMJG42-ptsP) were transformed into ATCC 17978 via electroporation to obtain ATCC 17978 gigAB and ATCC 17978 ptsP mutants. After tetracycline selection and sucrose counterselection, the clones were subjected to colony PCR. Gene deletions were confirmed by sequencing. For complementation of the deleted gigA/gigB, the entire open reading frames of gigA/gigB were amplified by PCR, cloned into pMJG120 or pMJG125 to obtain pMJG120-gigAB or pMJG125-gigAB, and transformed into ATCC 17978 gigAB via electroporation to obtain ATCC 17978 gigAB pMJG120-gigAB or ATCC 17978 gigAB pMJG125-gigAB.
ATCC 17978 gigAB was transformed with pMJG42-gigA/gigB to generate ATCC 17978' with in situ complementation of gigA and gigB. ATCC 17978 ptsP was transformed with pMJG42-gigAB to generate ATCC 17978 ptsP gigAB. All bacterial strains, plasmids, and primers in this study were summarized in Supplementary Tables 1-3.

Whole-Genome Sequencing
Eight strains of ATCC 17978 gigAB were randomly selected from different batches for whole-genome sequencing. Genomic DNA was prepared using the QIAamp DNA Mini Kit (Qiagen, Germany) and then subjected to whole genome sequencing (WGS) using the Illumina Hiseq2500 platform (Illumina, CA, United States) following the 2 × 100 bp protocol. The average sequencing throughput was 1 Gb. Raw fastq reads were trimmed by Trimmomatic for quality control (Bolger et al., 2014) and subsequently mapped against the reference genome of ATCC 17978-mff (Accession No. CP012004) with Bowtie2 (Langmead and Salzberg, 2012). Variant calling was performed using the bcftools call function with the default parameters (Danecek et al., 2021). We had submitted all of these data to NCBI BioProject database under the BioProject ID PRJNA738724. 1

Calculation of Gene Deletion Efficiency
After antibiotics selection and sucrose counterselection, 24 clones were randomly selected for colony PCR. Gene deletions were confirmed by sequencing. The gene deletion efficiency was calculated as (the number of the clones with successful deletion mutation)/24 × 100% The experiment was repeated three times, and data were expressed as the mean ± standard deviation (SD).

Efficiency of Plating Analysis
Overnight cultures of the indicated strains were back-diluted into fresh LB and grown for 2 h. After outgrowth, aliquots of the cultures were serially diluted. Then, a 10-µL aliquot was spotted onto LB agar plates with or without stressors as follows: HCl (medium adjusted to pH 5.5), ZnCl 2 (final concentration = 1.25 mmol/L). Colony forming units (CFU) were counted at 12 h after incubation at 37 or 50 • C. Efficiency of plating (EOP) was calculated as (CFU recovered on stress medium)/(CFU recovered on plain medium at 37 • C).
Each strain was grown overnight on the appropriate LB agar plate, and a single colony was picked and expanded in 2 mL LB broth overnight. A 1 µL aliquot was diluted at 1:1,000, and the dilution was added into triplicate wells of a 96-well plate at 200 µL/well. LB medium without bacteria was used as a blank. The OD 600 was determined every 15 min using a Biotek plate reader (Winooski, VT, United States). Growth curves were generated using GraphPad Prism 5 (San Diego, CA, United States). Each experiment was performed in triplicate and repeated three times. The mean was calculated for each experiment, and data were presented as the mean of three experiments.

Antibiotic Sensitivity Testing
Antibiotic sensitivity testing was performed as previously described (Gebhardt et al., 2015). The antibiotics used in this study are summarized in Table 1. Data were expressed as minimum inhibitory concentration (MIC).

Bacterial Killing Assay
Mouse BMDMs were plated in a 96-well plate at a density of 50,000 cells/well and cultured overnight. Cells were infected with wild-type ATCC 17978, ATCC 17978 gigAB, or ATCC 17978 gigAB pMJG120-gigAB at 5 × 10 5 CFU/mL. The plate was centrifuged at 2,170 rpm for 30 min at room temperature, followed by incubation at 37 • C for 30 min. After replacing the medium with RPMI 1640 containing 100 mg/L gentamicin to kill extracellular bacteria, the infected cells were incubated for an additional 1 h (t = 0 h). Then, the infected cells were cultured in RPMI 1640 supplemented with 25 mg/L gentamicin (wild type and gigAB strains) or 1.5 mg/L polymyxin ( gigAB pMJG120-gigAB strain). Cell lysates were collected at 0, 2, and 6 h post infection using phosphate buffered saline (PBS) containing 1% Triton-X100, serially diluted, and plated on LB agar plates. CFU were enumerated after 18 h of growth at 37 • C. Each experiment was performed in triplicate and repeated three times. The mean CFU of surviving bacteria was calculated for each experiment, and data were presented as the mean of three experiments.

G. mellonella Killing Assay
Infection of Galleria mellonella larvae (Knutson's LiveBait, Brooklyn, MI) was performed as described previously (Jacobs et al., 2014a;Gebhardt and Shuman, 2017). Briefly, the bacteria were grown overnight in an orbital shaker (37 • C, 200 rpm), and overnight cultures were resuspended in PBS to a final OD 600 of 1.0. G. mellonella larvae were randomly divided into three groups (n = 10/group). A total of 10 µL cultures (5 × 10 6 CFU/mL) were inoculated into the last left proleg of each larva. After injection, larvae were incubated at 37 • C. The number of dead larvae was recorded hourly. Each experiment was performed in triplicate and repeated three times. The mean larval survival was calculated for each experiment, and data were presented as the mean of three experiments.

Statistical Analysis
Data were expressed as the mean ± SD. Statistical analysis was performed using GraphPad Prism 5. Differences among groups were compared using one-way ANOVA followed by Dunnett's post-hoc test. Killing curves were plotted using the Kaplan-Meier method. A P-value of < 0.05 was considered statistically significant.

RESULTS
gigA/gigB Are Important for the Growth but Not Required for the Survival of ATCC 17978 In our preliminary study, we noticed that the gigA/gigB deletion efficiency in wild-type 17978 was only 4.2%, suggesting that loss of gigA/gigB inhibits the growth of 17978 ( Table 2). To further explore how the genetic background affects the efficiency of gigA/gigB deletion, we assessed gigAB mutation efficiency in various 17978 genetic backgrounds. All gene deletions and complementation were confirmed by sequencing. The results of these analyses are shown in Table 2, and indicate that strains harboring either a ptsP deletion or in trans-complementation of gigA/gigB greatly increased the frequency of isolating the gigA/gigB double deletion mutation, suggesting that ptsP deletion and gigA/gigB complementation can compensate for the apparent growth defect caused by loss of gigA/gigB. In the arabinose-inducible pMJG125 vector-based complementation of gigA/gigB background, we observed that the gigAB colonies were smaller than wild-type colonies in the absence of arabinose; this phenotype was eliminated by the supplementation with 1% arabinose ( Table 2 and Supplementary Figure 1). This finding further confirms that gigA/gigB are important for the growth of 17978 and that complementation of gigA/gigB with arabinose supplementation promotes the growth of gigAB mutant to the wild-type level.
To determine if the loss of gigA/gigB required the generation of suppressing mutations, we performed whole genome sequencing on eight 17978 gigAB clones isolated from different batches of gene knockout experiments. We found that, other than the gigA/gigB deletion, the genome of each of the sequenced gigAB clones was 100% identical to the genome of ATCC 17978-mff reference strain, suggesting that deletion of the gigA/gigB genes does not require suppressing/compensatory mutations and that ATCC 17978 can survive without gigA/gigB. Thus, gigA/gigB are important for the growth but not required for the survival of ATCC 17978 under our routine laboratory culturing conditions.

Loss of ptsP and/or gigAB Does Not Affect Antibiotic Resistance of ATCC 17978
To explore the roles of ptsP and gigA/gigB in the antibiotic resistance of ATCC 17978, we performed antibiotic susceptibility tests in the wild type and gene deletion strains. As shown in Table 1, although the MIC of apramycin, chloramphenicol, and kanamycin were decreased in at least two deletion mutation strains compared with those in wild-type ATCC 17978, the results did not reach statistical significance. These data suggest that, in contrast to our previous findings in the A. baumannii AB5075 strain background, ptsP and gigA/gigB are not required for antibiotic resistance of ATCC 17978.

Loss of ptsP Restores the Growth of 17978 gigAB to the Wild-Type Level
To explore the involvement of ptsP in gigA/gigB-mediated growth of ATCC 17978, we performed growth curve analyses. As shown in Figure 1A, 17978 gigAB exhibited remarkably suppressed growth compared with the wild-type, whereas 17978 ptsP exhibited a comparable growth rate to the wild-type, suggesting that gigA/gigB contribute to 17978 growth. Interestingly, 17978 ptsP gigAB showed comparable growth to the wild-type strain, indicating that loss of ptsP alleviates the growth defect associated with the loss of gigA/gigB. In addition, pMJG125based complementation of gigA/gigB also restored the growth of 17978 gigAB to the wild-type level in the presence of arabinose ( Figure 1B).

gigA/gigB Mediate in vitro High-Temperature Resistance of ATCC 17978
We next sought to explore any additional roles of gigA/gigB and ptsP in stress resistance of ATCC 17978. Although the wildtype 17978 grown at 50 • C showed a moderately reduced colony size phenotype when compared with those grown at 37 • C, no significant loss of CFU was observed (EOP = 1; Figure 2A, left panel). However, loss of both gigA and gigB resulted in a dramatic reduction in CFU at 50 • C (EOP = 10 −5 ; Figure 2A, right panel), suggesting that gigA and gigB contribute to hightemperature resistance of 17978. In the absence of arabinose, complementation of both gigA and gigB partially restored the growth of gigAB mutant at 50 • C (EOP = 10 −3 ; Figure 2B, left panel). Importantly, arabinose supplementation further restored the growth of gigAB mutant with gigA/gigB complementation to the wild-type level at 50 • C (EOP = 1; Figure 2B, right panel), despite the small sizes of the colonies. This finding suggests that gigA/gigB contribute to high-temperature resistance of 17978 on solid media. As observed in the growth curves described above, 17978 ptsP gigAB and 17978 ptsP exhibited comparable growth at 50 • C (EOP = 1; Figure 2C), suggesting that loss of ptsP restores the growth of gigAB mutant under high-temperature  stress. Taken together, these results suggest that gigA/gigB mediate high-temperature resistance of 17978 on LB agar plates, whereas ptsP negatively regulates this response. When we examined the ability of the gigAB strain to survive acid stress (pH = 5.5), we did not observe significant differences in the growth between the wild-type and gigAB mutant strains (EOP = 1; Figure 3A), suggesting that the 17978 strain is not sensitive to pH stress as measured herein, and that the loss of gigA and gigB does not confer an acid stress sensitivity on the 17978 strain.
When cultured on LB containing Zn 2+ , both wild-type and gigAB mutant demonstrated significantly suppressed growth compared with those cultured on LB without Zn 2+ (EOP = 10 −4 , Figure 3B). No major difference was observed in the growth between the wild-type and gigAB mutant. This finding suggests that factors other than gigA and gigB mediate zinc resistance of ATCC 17978.

gigA/gigB Protect ATCC 17978 From BMDM Killing
Evading macrophage phagocytosis is critical for the survival of pathogens in vivo (Rosales and Uribe-Querol, 2017). To investigate the roles of gigA/gigB in macrophage killing evasion of ATCC 17978, we infected murine BMDMs with the wildtype, gigAB mutant, and gigAB complementation strains and monitored their survival and replication. When BMDMs were infected with wild-type 17978, we observed a 10-fold reduction of intracellular live bacteria at 2 h after infection (Figure 4). On the other hand, when BMDMs were infected with gigAB mutant, we observed a 300-fold reduction of live bacteria in BMDMs at 2 h after infection (Figure 4), suggesting a decreased replication ability of the gigAB mutant. Importantly, the gigAB pMJG120-gigAB complementation strain exhibited a similar trend of survival and replication and comparable CFU at different time points to the wild-type 17978 (Figure 4). These results suggest that gigA and gigB promote macrophage killing evasion of ATCC 17978.

gigA/gigB Are Required for Killing G. mellonella
To examine the roles of gigA/gigB in the virulence of ATCC 17978, we performed a G. mellonella killing assay. As shown in Figure 5, inoculation of G. mellonella larvae with wild-type 17978 resulted in a rapid killing of the larvae starting 8 h after inoculation. No killing was observed in the larvae that received gigAB mutant within 48 h after inoculation. Complementation of both gigA and gigB restored the virulence of bacteria to nearly  wild-type level. Thus, gigA/gigB are required for the virulence of ATCC 17978. Much like for the growth and temperature studies described above, both the 17978 pstP and 17978 ptsP gigAB strains killed larvae with similar kinetics as the wild-type 17978 strain, suggesting that the loss of ptsP restores the virulence defect caused by the gigAB deletion.

DISCUSSION
In this work, we sought to investigate the roles of gigA/gigB in the survival, stress resistance, macrophage killing evasion, and virulence of A. baumannii ATCC 17978 as well as the involvement of ptsP in gigA/gigB signaling. We found that gigA/gigB are important for growth of A. baumannii ATCC 17978, but are not explicitly required for survival of 17978. Indeed, the gigAB mutant strain exhibited growth defects at both 37 • C and 50 • C compared with the wild-type strain, which was effectively restored by pMJG125-based gigA/gigB complementation in the presence of arabinose or loss of ptsP. Furthermore, gigA/gigB protected 17978 from murine BMDM killing and were required for the virulence of 17978 in G. mellonella. Bacterial genetics remains an important and powerful tool for revealing the function(s) of specific genes. Efficient construction of gene knockouts or other types of mutations in bacteria often requires modifications of genetic background (Xu and Zhang, 2016). Our preliminary data have shown that FIGURE 4 | Bone marrow-derived macrophage killing of wild-type and mutant ATCC 17978. Murine bone marrow derived macrophage (BMDMs) were cultured for 24 h, then challenged with wild-type ATCC 17978, ATCC 17978 gigAB, or ATCC 17978 gigAB pMJG120-gigAB at 5 × 10 4 colony forming unit count (CFU)/mL in the presence of 1 mM IPTG. BMDMs were lysed at 0, 2, or 6 h after challenge and surviving bacteria were quantified via standard plate count method.
the gigA/gigB deletion efficiency in wild-type 17978 was only 4.2%, suggesting that gigA/gigB are critical for the survival of ATCC 17978. We further found that gigA/gigB complementation or ptsP deletion significantly improved gigA/gigB deletion efficiency, suggesting that gigA/B complementation or ptsP deletion compensates for the lack of gigA/B in 17978. This is consistent with our previous study showing that loss of ptsP in the A. baumannii gigA or gigB mutant restores the growth of A. baumannii in G. mellonella larvae (Gebhardt and Shuman, 2017).
When studying genes essential for bacterial growth and/or survival, it is not uncommon to inadvertently isolate clones which harbor compensatory or suppressing mutations that alleviate the phenotype of the particular genes being studied (MacLean and Vogwill, 2014). To exclude the possibility that the gigAB strain acquired such compensatory mutations, we performed whole genome sequencing in multiple independently derived gigAB clones and found that, with the exception of the gigA/gigB deletion, the genome of gigAB clones displayed 100% identity to the genome of the wild-type strain, suggesting that gigA/gigB deletion does not require subsequent compensatory mutations, further confirming that 17978 can survive without gigA/gigB.
When we knocked out gigA/gigB in the genetic background of pMJG125-gigAB conditional strain, we observed that in the absence of arabinose, the colonies of gigAB mutants were smaller than those of the wild-type ( Table 2 and Supplementary  Figure 1). In addition, gigAB mutant still showed growth defect even after the complementation of gigA/gigB in the absence of arabinose (Figure 1). Of note, arabinose supplementation effectively reversed these effects. We attribute these observations to leaky basal expression from the arabinose-promoter on the multi-copy pMJG125 plasmid.
In addition to the growth in LB medium, we also investigated the roles of gigA/gigB in ATCC 17978 in response to several environmental stresses, including antibiotics, high temperature, Zn 2+ , and acid. Neither the wild-type nor the gigAB mutant showed significant growth defect to acid stress ( Figure 3A). Additionally, we did not observe significant differences for MIC values for various antibiotics ( Table 1) and colony formation in the presence of Zn 2+ (Figure 3B) between the wild-type and the mutant strain lacking both gigA and gigB. These results suggest that factors other than gigA/gigB regulate the responses of ATCC 17978 to antibiotics and Zn 2+ stresses, in contrast to what was previously observed in the more virulent AB5075 strain (Gebhardt et al., 2015;Gebhardt and Shuman, 2017;Blaschke et al., 2018). For example, it has previously been reported that the chromosomally-encoded efflux pump CraA, AdeAB efflux system, and incubation temperature regulate antibiotic resistance of ATCC 17978 (Adams et al., 2018;De Silva et al., 2018;Kroger et al., 2018). Additionally, transcriptional analyses have shown that zinc resistance efflux pumps are responsible for zinc FIGURE 5 | Killing of G. mellonella larvae. G. mellonella larvae were inoculated with 5 × 10 6 CFU/mL of wild-type ATCC 17978, ATCC 17978 gigAB, or ATCC 17978 gigAB pMJG120-gigAB (n = 10 larvae/group). After injection, larvae were incubated at 37 • C. The number of dead larvae was recorded hourly. stress response in ATCC 17978, including two cation diffusion facilitator transporters, one heavy metal efflux transporter, and one P-type ATPase (Hassan et al., 2017). That there are differential consequences of gigA/gigB deletion in the AB5075 background (i.e., aminoglycoside and zinc sensitivity) and the ATCC 17978 background (i.e., growth defect under routine culture conditions) suggests that some of the inputs and/or outputs of the GigA/GigB signaling pathway have diverged since the two strains separated; yet, other facets of the pathway, such as growth at elevated temperature and virulence, have remained intact. Further research will be required to understand the molecular mechanisms that underlie the different stress responses that are regulated by GigA/GigB amongst these two isolates.
Of note, our results showed that complementation of the gigAB deletion strain with a plasmid-borne copy of gigA/gigB restored growth on agar plates at high temperature and that a subsequent deletion of ptsP in the gigAB background also alleviated the high temperature growth defect caused by the loss of both gigA and gigB (Figure 2), consistent with previous observations in the AB5075 strain (Gebhardt and Shuman, 2017).
We finally examined the roles of gigA/gigB in evading macrophage phagocytosis and killing G. mellonella larvae. Our data indicate that gigA/gigB are required for 17978 in killing G. mellonella: no larvae died within 48 h after inoculation with gigAB mutant. Additionally, we found that gigA/gigB contribute to the macrophage killing evasion of ATCC 17978, as evidenced by the decreased intracellular live bacteria and the suppressed bacterial replication in murine BMDMs infected with gigAB mutant compared with those infected with the wild-type strain ( Figure 5). As it has been reported that RNA chaperone Hfq and superoxide dismutase of ATCC 17978 also play important roles in evading macrophage phagocytosis (Heindorf et al., 2014;Kuo et al., 2017), it will be interesting to examine if the loss of gigA and/or gigB leads to altered expression of these virulence factors.

CONCLUSION
In this study, we demonstrate that gigA/gigB are important for the growth of A. baumannii strain ATCC 17978, although they are not explicitly required. The gigAB mutant exhibits growth defects at both 37 and 50 • C, which can be restored either through gigA/gigB complementation or by loss of ptsP. In contrast to findings in the A. baumannii AB5075 background (Gebhardt and Shuman, 2017), gigA/gigB do not appear to alter the response of strain 17978 to antibiotics or Zn 2+ stress. Finally, like strain AB5075, the gigA/gigB genes are required for the virulence traits of strain ATCC 17978 in both resisting killing by macrophage and the G. mellonella infection model.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: http://www.ncbi.nlm. nih.gov/bioproject/, PRJNA738724.

ETHICS STATEMENT
The animal study was reviewed and approved by the Ethics Committee of the University of Chicago Medical Center.

AUTHOR CONTRIBUTIONS
HZ performed the all stepss of experiment, analyzed the experimental data, and drafted the manuscript. MG helped construct A. baumannii ATCC17978 mutants. DC helped perform the BMDM isolation and bacterial killing experiments. YY analyzed whole genome sequences and helped to analyze data. MG and DC revised the manuscript. HS designed the study and revised the manuscript. All authors read and approved the final manuscript.

FUNDING
This work was supported by a research grant from the National Natural Science Foundation of China (81971897) and a research grant from the Natural Science Foundation of Zhejiang Province LQ20H0006). The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.