ABSTRACT
Hospital environments serve as excellent reservoirs for the opportunistic pathogen Acinetobacter baumannii in part because it is exceptionally tolerant to desiccation. To understand the functional basis this trait, we used transposon sequencing (Tn-seq) to identify genes contributing to desiccation tolerance in A. baumannii strain AB5075. We identified 142 candidate desiccation tolerance genes, one of which encoded the global post-transcriptional regulator CsrA. We characterized CsrA in more detail by using proteomics to identify proteins that were differentially present in wild type and csrA mutant cells. Among these were a predicted universal stress protein A, an iron-containing redox protein, a KGG-domain containing protein, and catalase. Subsequent mutant analysis showed that each of these proteins was required for A. baumannii desiccation tolerance. The amino acid sequence of the KGG-domain containing protein predicts that it is an intrinsically disordered protein. Such proteins are critical for desiccation tolerance of the small animals called tardigrades. This protein also has a repeat nucleic acid binding amino acid motif, suggesting that it may protect A. baumannii DNA from desiccation-induced damage.
INTRODUCTION
Hospital-acquired infections are an important healthcare concern and economic burden (1, 2) and environmental persistence plays a critical role in the transmission of bacteria that cause these infections (3–6). One such bacterium is Acinetobacter baumannii, an opportunistic pathogen that infects very sick patients. It is responsible for about 2% of nosocomial infections in the United States and Europe and the frequencies are higher in the rest of the world. A. baumannii is especially problematic because on a global basis, about 45% of isolates are multi-drug resistant (7). A factor that contributes to the prevalence of A. baumannii in hospital settings is desiccation tolerance. A. baumannii can survive in a desiccated state on inanimate dry surfaces for days to several months (8–10). These surfaces include materials that are often encountered in the hospital, such as polyvinyl chloride, rubber, and stainless steel (11).
When desiccated, bacteria must respond to diverse stresses that include accumulation of reactive oxygen species, loss of cytoplasmic volume, and loss of cell membrane integrity (12, 13). Proteomics analysis of A. baumannii showed that desiccated cells had higher levels of proteins involved in protein stabilization, antimicrobial resistance, and reactive oxygen species detoxification (14). Attributes of A. baumannii, that have been shown to be associated with desiccation tolerance include biofilm formation (15, 16) and protein aggregation (17). LpxMAB-dependent acetylation of lipid A is essential for survival of A. baumannii ATCC17978 at 40% humidity (18), and a recA mutant of ATCC17978, defective in DNA repair, had pleiotropic effects, including a defect in desiccation tolerance (19). katE, encoding catalase also contributes to desiccation tolerance (20). To further probe the functional basis for desiccation tolerance in A. baumannii we applied transposon sequencing (Tn-seq), an unbiased, high-throughput genetic screening approach, to generate a comprehensive list of genes that may be important for desiccation survival. From 142 candidate genes identified in the Tn-seq screen, single mutant analysis of a small subset revealed six genes that likely contribute to desiccation tolerance, one of which was csrA (ABUW_2750). csrA encodes a global post-transcriptional regulator found in gamma-proteobacteria and was recently reported by another group as important for desiccation tolerance of A. baumannii (21). Here we expanded on this recent report and identified CsrA-controlled proteins, several of which turned up in the Tn-seq screen, that play a role desiccation survival.
RESULTS
Desiccation assay
Previous studies have shown that A. baumannii can survive in a desiccated state for days to several months (8–11, 20). For these and other desiccation studies, investigators worked with a variety of strains and usually incubated cells at either 30% relative humidity (RH) or in room air, which varied between 25 and 61% RH in one study (20). These differences can make it difficult to compare desiccation phenotypes between studies. Thus we thought it important to establish a robust desiccation assay that reduces experimental variables like choice of strains, drying times, and RH during desiccation.
Following from previous reports, saturated calcium chloride hexahydrate solution placed in a sealed plastic Snapware container caused the RH inside the container to rapidly equilibrate to 30% (9). We found that use of DRIERITE instead of calcium chloride, resulted in an RH of 2%. To test desiccation tolerance, we grew bacteria to a desired density in tryptone-yeast extract (TY) broth, harvested them, washed them twice with phosphate buffer, and resuspended them in buffer to a final OD600 of 1. Drops of cell suspension were placed on polycarbonate membranes and filtered to allow for rapid drying. The membranes were placed in uncapped 15 ml conical centrifuge tubes and incubated in desiccation containers. After various periods of incubation, buffer was added to each centrifuge tube followed by 5 min of shaking on a rotary shaker. Viable cell numbers were then determined by plating on TY agar. To control for the stress of filtration we did viable counts immediately following filtration and took this as our “day 0” time point.
Relative desiccation tolerance of A. baumannii strains
As shown in Fig 1A, A. baumannii strain AB5075 and Escherichia coli strain W3100 each survived desiccation at 30% RH far better than Pseudomonas aeruginosa strain PAO1. However at 2% RH, A. baumannii survived far better than either E. coli or P. aeruginosa. As has been reported (20, 22), we found that A. baumannii stationary phase cells were much more tolerant to desiccation than actively growing cells (Fig. S1) and so we routinely used stationary phase cells in our desiccation assays. We also tested the desiccaton tolerance of two additional frequently used laboratory strains of A. baumannii, ATCC17978 and ATCC19606, and found that they did not survive well when desiccated at 2% RH for six days (Fig. 1B). However, these strains survived when the RH was 30%, which is similar to the RH of many hospital environments. Strain-to-strain variation in desiccation tolerance has previously been reported (10, 20).
In our desiccation experiments we wanted to use a condition under which A. baumannii survived better than E. coli and we also wanted to provide strong selection pressure in our screen for genetic determinants of desiccation tolerance. To satisfy these requirements we used A. baumannii strain AB5075 and 2% RH in subsequent experiments. This strain was isolated from a surgical wound, is multidrug resistant and is highly virulent in an animal model (23). A comprehensive ordered mutant library of AB5075 is available that has two to three seqenced Tn insertions in each gene and is called the three-allelle library (24).
Unbiased screening to identify desiccation sensitive mutants by Tn-seq
To begin to define genetic mechanisms underlying desiccation tolerance in A. baumannii, we created a saturating high-density transposon mutant pool containing approximately 400,000 individual A. baumannii mutants. We grew the transposon mutant pool to stationary-phase and plated cells on TY medium at day 0 and day 6 of desiccation at 2%RH. After a brief period of growth, cells were harvested, pooled and processed for mapping of Tn insertions. The transposon mutant pool survived desiccation as well as wild-type strain AB5075, indicating that the transposon itself did not affect desiccation tolerance (Fig. S2).
Excluding genes that were not well-represented (less than100 Tn reads/kb) in the day 0 sample, we found that 142 genes showed a 2.5-fold or more decrease in abundance of Tn insertions after 6 days of desiccation (Table S1). We picked 12 genes that had a 10-fold or more decrease in transposon insertions after a 6-day period of desiccation to validate their desiccation phenotypes. When possible, we tested two different transposon mutants (transposon insertions in different positions of the gene) for each of these 12 genes from the three-allele library. A. baumannii AB5075 produces opaque and translucent colony variants that interconvert at high frequency and reflect changes in the thickness of capsular exopolysaccharide (25). AB5075 cells with decreased capsule production were as much as 100-fold more sensitive to desiccation (26). Here, we used only opaque colonies of AB5075 and its mutant derivatives in our desiccation assays. We found that when mutated, six of the 12 genes we tested had 2-fold or greater defects in desiccation tolerance (Table 1). We were interested to see that one of the genes was csrA. CsrA, also called RsmA in some bacteria, is an important global regulator of mRNA translation in gamma proteobacteria. It has diverse effects on many processes including motility, biofilm formation, quorum sensing and secretion of virulence factors, depending on the species (27–30).
CsrA is critical for desiccation tolerance
csrA mutants from the three-allele library were only two-fold more sensitive to desiccation than the wild type. To try to confirm its importance for desiccation tolerance we constructed a csrA deletion mutant (ΔcsrA). We found that the ΔcsrA strain grew poorly on TY agar and had an elongated cell morphology when grown in TY broth (Fig. 2A). On agar plates, large colonies frequently appeared on a backgound of poor growth, likely due to occurance of second site suppressor mutations in the ΔcsrA strain. The ΔcsrA strain was also defective in growth on other nutrient-rich media, including Luria broth, nutrient broth, and tryptone soy broth. A similar sensitivity to growth in complex media was reported by Farrow et al for a several A. baumannii strains including strain AB5075 (21). In agreement with Farrow et al., a ΔcsrA mutant grew as the wild type in defined medium, in our case, M9 minimal medium with 10 mM succinate as a sole carbon source (M9/succinate), and it had close to a wild type cell morphology (Fig 2A). A Yersinia enterocolitica csrA mutant, has a growth defect in LB due to the presence of 90 mM of NaCl (31). However, the A. baumannii ΔcsrA mutant was not sensitive to NaCl. In fact, the mutant grew in M9/succinate supplemented with up to 100 mM of NaCl without a significant reduction of growth compared to the wild type.
When desiccated after growth in M9/succinate to stationary phase, the ΔcsrA mutant lost almost all viability over 6 days (Fig. 2B and Table 1). The desiccation phenotype was complemented by expressing csrA in trans. ΔcsrA mutant cells incubated for 6 days after being filtered and resuspended in PBS remained fully viable (Fig 2B). As we were preparing this paper for publication, Farrow et al reported that csrA mutants of A. baumannii strains AB09-003 and ATCC 17961 were sensitive to desiccation in room air over a period of 14d. CsrA in these strains was also required for biofilm formation and virulence in a Galleria melonella infection model (21).
CsrA affects mutiple cellular processes in A. baumannii
To identify genes whose translation might be regulated by CsrA, we compared the proteomes of wild-type and ΔcsrA cells (Table S3A). There were 97 proteins present at higher levels in the ΔcsrA mutant compared to the wild type (ratio of ΔcsrA/WT ≥2.5, Table S3B). Among these were proteins for type IV pilus assembly, synthesis of the siderophore ferric acinetobactin, and a glutamate/aspartate transporter. The ΔcsrA mutant also had elevated levels of enzymes for for the catabolism of hydroxcinnamates, phenylacetate and quinate. Levels of an alcohol dehydrogenase (ABUW_1621) and an aldehyde dehydrogenase (ABUW_.1624) were also elevated. The ΔcsrA mutant was defective in pilus-mediated twtiching motility as assessed by movement aross a soft-agar plate (Fig. 3A). The mutant also had a severe growth defect when grown on succinate in the presence of ethanol (Fig. 3B). One possible explanation for this is that the csrA mutant metabolized ethanol to form toxic acetaldehyde to levels that slowed growth, and insufficient aldehyde dehydrogenase activity was present in cells to relieve this toxicity.
There were 106 proteins present in lower amounts in the ΔcsrA mutant compared to the wild type (ratio of WT/ΔcsrA ≥ 2.5, Table S3C). A large proportion of these (39%) are annotated as hypothetical proteins. Several membrane proteins, and proteins annotated as involved in β-lactam antibiotic resistance (ABUW_1194, 2619, and 3497), trehalose synthesis (ABUW_3123) and possibly biofilm formation (ABUW_0916) were in lower abundance in the ΔcsrA mutant compared to wild type. As reported previously, a ΔcsrA mutant did not form biofilms, (Farrow 2020) and this phenotype was complemented by expressing csrA gene in trans (Fig. 3C). The ΔcsrA proteome profile also suggested that CsrA was invovled in promoting the expression of proteins invovled in oxidative stress, including peroxidase (ABUW_0628) and catalase (katE, ABUW_2436). When tested for catalase activity, we found that the ΔcsrA mutant lacked this trait (Fig. 3D).
Genes important for desiccation tolerance in A. baumannii AB5075
We took advantabge of the three-allele transposon library to test how important some of the gene transcripts that were likely to be controlled CsrA were for desiccation tolerance. All the genes that we found to possibly be important for desiccation tolerance are listed in Table 1. The results of desiccation assays for all the mutant strains that we tested in this study are listed in Table S2. katE, and ABUW_2639 mutants were about 5-fold more sensitive to desiccation than the wild type, whereas ABUW_2433 and ABUW_2437 mutants were greater than 100 fold more sensitive to desiccation than the wild type (Table 1, Fig 4). The phentoytpes of ABUW_2433 and ABUW_2437 mutants could be complemented (Fig S3). The ABUW_2433 protein has 411 amino acids and is annotated as a KGG domain-containing protein. The KGG domain comprises a small region in the N-terminus of the protein and the remainder of the protein is annotated by InterPro as a disordered region that includes a series of AT_hook DNA binding mofits (SMART SM00384). The full length ABUW_2433 sequence was predicted to be intrinsically unstructured when queried with the IUPred3 tool (https://iupred.elte.hu) (32). ABUW_2437 is annotated as an iron-containing redox enzyme or a heme-oxygenase -like protein (Fig 4). The predicted ABUW_2437 transcript has traits characterisitic of a target of CsrA post-transcriptional regulation. The DNA sequence predicts a relatively long (316 bp) 5’ untranslated region (316 bp) and there is a predicted CsrA binding motif (GGA) in the ribosome binding site of the transcript. ABUW_2639 is annoated as belonging to a universal stress protein A family. It has been shown to protect A. baumannii ATCC17987 from oxidative stress of hydrogen peroxide (33).
We wondered if ABUW_2433 and ABUW_2437 might play a role in promoting desiccation tolerance of the two A. baumannii strains, ATCC17978 and ATCC19606, that do not survive well at 2% RH (Fig 1B). ATCC19606 has the gene region shown in Fig 4 intact, but the gene that is homologous to ABUW_2433, encoding the KGG domain-containing protein, is annotated as a pseudogene. ATCC 17978 appears to be missing a gene in homologus to ABUW_2433. However it has conitguous katE and iron-containing redox protien genes (A1S_1386 and A1S_1385). Expression of the two AB5075 genes in trans improved the survival of the two ATCC strains at 2% RH (Fig 5), providiing evidence that ABUW_2433 and ABUW_2437 are generally important for desiccaton tolerance.
Other possible desiccation tolerance genes
As shown in Table 1, we identified an additional five genes, some but not all of which are likely regulated by CsrA, that may have a small role in desiccation tolerance. otsA, encoding tehalose-6-phosphate synthase, is the only one of the five for which we can hypothesize some connection to desiccation. Trehalose has been shown to play a significant role in desiccation tolerance of eukaryotes and bacteria (34, 35) and trehalose added extrinsically to cultures increased the desiccation tolerance A. baumannii ATCC 19606 (22). However, a ΔmtlD-otsB mutant of ATCC19606, defective in endogenous production of the compatible solutes, mannitol and trehalose, was not more sensitive to desiccation than the wild type (22).
DISCUSSION
Depletion of water during desiccation leads to loss of membrane integrity and accompanying disruption of aerobic respiration results in the generation of reactive oxygen species, including hydrogen peroxide (36). That katE, encoding catalase, contributes to desiccation tolerance makes sense in this context. Proteomics analyses of A. baumannii showed that proteins involved in redox defense including catalase, alkyl peroxidase reductases and superoxide dismutase were elevated in stationary-phase cells (37), which is consistent with the observation made by many that cells stationary-phase cells survive desiccation much better than exponentially growing cells.
Since the desiccation -tolerance genes ABUW_2433 and ABUW_2437 are near or adjacent to katE, it seemed important to consider that they might somehow mediate oxidative stress tolerance even though the amino acid sequences of the encoded proteins don’t have motifs typically associated with reactive oxygen species detoxification. However, we were unable to demonstrate that ABUW_2433::Tn and ABUW_2437::Tn mutants were sensitive to hydrogen peroxide, nitrous oxide or paraquat - all powerful oxidizing agents. In addition, a study that looked at effects of hydrogen peroxide exposure on gene expression in A. baumannii, found that katE but not ABUW_2433 or ABUW_2437, was expressed at elevated levels and neither of these genes is part of the OxyR regulon that controls the response to oxidative stress in A. baumannii (38).
The physical properties and cellular function of ABUW_2433 will be fascinating to explore. It is an intrinsically disordered protein that is highly hydrophilic, with 27% positively charged amino acids residues and 31% negatively charged residues. It is also predicted to assume a collapsed or extended conformation, likely depending on its context (ROBETTA PFRMAT TS prediction; https://robetta.bakerlab.org). ABUW_2433 has 13 repeated AT-hook DNA binding motifs that occupying about 70% of the protein. This motif preferentially binds to AT-rich sequences in the minor groove of DNA. AT-hook DNA binding motifs are found primarily in eukaryotic proteins, many of which have roles in transcriptional regulation (39, 40). Only 8.5% of annotated AT hook DNA binding motifs are found in bacteria, but about half of these are found in gamma proteobacteria, the group to which A. baumannii belongs. We hypothesize that ABUW_2433 binds to A. baumannii DNA and somehow protects it from desiccation-induced damage. IDPs are critical for the microscopic animals called tardigrades to survive desiccation. When desiccated, some of these proteins vitrify and probably trap desiccation sensitive molecules in a noncrystalline amorphous matrix, thereby protecting them from denaturation or other forms of destruction (41, 42). IDPs or proteins with intrinsically disordered regions are less common in prokaryotes than in eukaryotes, but drawing from work on eukaryotes, they have been proposed to play a central role in cellular process in bacteria that may depend on the formation of molecular condensates (43). It is possible that this is important for the viability of desiccated A. baumannii.
Although not much work has been done on Acinetobacter CsrA, based on what is known for other gamma proteobacteria, we hypothesize that a set of ncRNAs that is induced by a GacSA (ABUW_3306 and ABUW_3639) two -component regulatory system, controls the repressor activity of CsrA by sequestering it (44). We can draw a link between the GasSA system and CsrA because they both appear to control catabolism of the aromatic compound phenylacetate. An A. baumannii ΔgacA mutant is unable to catabolize phenylacetate (45), and our proteomics results suggest that CsrA acts to repress the synthesis of at least one enzyme required for phenylacetate degradation. We hypothesize that a ΔgacA mutant does not synthesize ncRNAs that would normally “sponge-up” CsrA, thus allowing CsrA to bind to the 13 -gene phenylacetate mRNA transcript to repress its translation. At this point we do not have a clear understanding of the inventory of A. baumannii ncRNAs that may bind to CsrA, but ncRNAs are abundant in AB5075, with several of them expressed at extremely high levels (46). The desiccation phenotype of CsrA appears to depend on its ability to activate translation and although it’s difficult to reconcile this activity with a model where CsrA is sequestered by ncRNAs, it is known that ncRNA turnover can occur resulting in the release of free CsrA (29). Most of what is known about mechanisms of CsrA action centers on its role as a repressor of translation (30, 47–49) and it may be of interest to probe its capability as an activator in A. baumannii.
We found that A. baumannii AB5075 survived desiccation for six days at 2% RH much better than two other A. baumannii strains that we tested, but it is important to note that most studies of desiccation tolerance have been carried out at about 30% RH or in room air and the emphasis has been on the number of days or months that a particular strain remains viable when desiccated. When Farrow et al (20)tested the survival of several strains that were dried and incubated at a relative humidity of 25–61% (mean 46%) they found AB5075 to have an average survival time of 90 days, whereas strains ATCC19606 and ATCC17978 had average survival times of 3 and 34 days respectively. Even though AB5075 is tolerant to desiccation over months at a mean RH of 46%T and over days at 2% RH, we cannot necessarily conclude that the same sets of genes are needed for desiccation tolerance under these two different conditions. For example, Farrow et al. (20) found that the response-regulator protein BmfR was important for desiccation tolerance of ATCC17978 in long term desiccation assays, whereas we did not observe a role of bmfR in protecting AB5075 from desiccation in shorter term incubations at 2%RH (Table S2).
The emergence of A. baumannii is great threat in healthcare facilities worldwide, and there is an urgent need for development of new antibiotics and new strategies for infection control and prevention. Since CsrA is involved in several clinically important traits, including desiccation tolerance, biofilm formation, and pathogenesis, the development of modulators of CsrA-RNA interactions may have potential for improving the efficacy of biocides or for treatment of A. baumannii infections (50).
MATERIALS AND MESTHODS
Bacterial strains and growth conditions
Strains used in this study are listed in Table S4A. Strain AB5075 (AB5075-UW) was used as a wild type (24) and individual transposon mutants were obtained from the Manoil lab comprehensive ordered transposon mutant library at the University of Washington (24). All strains except for the ΔcsrA mutant were routinely grown and maintained in TY (10 g Tryptone, 5 g Yeast extract, and 8 g NaCl in 1000 ml) medium or BBL Trypticase Soy Broth (TSB) media at 37°C, unless otherwise stated. The ΔcsrA mutant was grown in M9/succinate.
Desiccation assay
Strains from a frozen stock (−80°C ) were streaked onto TY plates and incubated at 37°C. Colonies (three to five) were picked and inoculated into 2 ml of TSB, and cultures were grown overnight at 37°C with a shaking speed of 200rpm. Overnight cultures were diluted to yield an initial OD600 of 0.025 in 10 ml TSB in a 50 ml Erlenmeyer flask. Cultures were grown at 37°C with a shaking speed of 200 rpm to mid-exponential-phase (OD600=0.4 to 0.6) or stationary-phase (24 hours after inoculation). Cells were harvested by centrifugation and washed twice with Dulbecco’s phosphate-buffered saline (DPBS, Gibco), and cell density was adjusted to OD600=1 (about 5 x 108 cells/ml) with DPBS. Cell suspension (2 spots of 50 μl each per membrane) was filtered onto a 0.4 μm Whatman nucleopore polycarbonate track-etched membranes (25 mm diameter) that had been placed in Nalgene analytical filter unit, and the membranes were then placed into 15 ml uncapped centrifuge tubes. To obtain the T0 (baseline) viable cell number, 1 ml of DPBS was immediately added to one tube and incubated for 5 min at room temperature (24 ± 2°C) on a rotary shaker. Viable cell numbers were determined by plating on TSB agar. For desiccation, tubes with membranes were placed in a Snapware containers (2.3 x 6.3 x 8.4 inches) that contained DRIERITE in the lids of 50 ml centrifuge tubes (x4, 7.5 g of DRIERITE desiccant in each lid) or saturated calcium chloride hexahydrate solution in 5 ml beaker (x8) to yield the RHs of 2% or 30% (± 2), respectively. The Snapware containers were incubated at room temperature. Digital hygrometers (VWR International Ltd) were placed in each container to monitor the RH. At desired time points, tubes containing membranes were removed from the containers, 1 ml of DPBS was added to each tube, and incubated for 5 min at room temperature on a rotary shaker. Viable cell counts were determined on TSB agar. For each strain, a minimum of three biological replicates of desiccation assays were performed except for individual transposon mutants, which were assayed twice for each allele.
Screening of desiccation sensitive mutant by Tn-seq
We created a high-density transposon mutant pool containing approximately 400,000 A. baumannii mutants by combining two separately constructed transposon mutant pools. The first transposon mutant pool was previously constructed and selected on LB agar (24) The second transposon mutant pool was constructed on Neidhardt MOPS minimal medium (51) excluding NaCl, and sodium succinate was used as a sole carbon source as follows: T26 mutagenesis was carried out as previously described (24), except following electroporation, cells were incubated in 1 ml of minimal medium with rolling at 37°C for 2 hours. Cells were flash frozen in 5% dimethyl sulfoxide (DMSO) and stored at −80°C for later selection. For selection, frozen cell aliquots were thawed on ice, pelleted, re-suspended in 1 ml fresh minimal medium, and plated onto a Q-tray (Genetix) containing 250 ml minimal medium, 1.2% agarose, and 12.5 μg/ml tetracycline (Tc). After incubating for 16 h at 37°C, cells were scraped, resuspended in 3.5 ml of minimal medium supplemented with 10% glycerol, and aliquots were flash frozen and stored at −80°C. This yielded a density of approximately 1,000 to 2,000 transposon mutants per Q-tray. We repeated these procedures a total of 69 times, and all 3.5 ml aliquots were thawed and mixed to create the final transposon mutant pool, which resulted in a total of approximately 120,000 individual transposon mutants from minimal medium. Finally, 1 ml aliquots of the final transposon mutant pool were flash frozen and stored at −80°C.
Aliquots of the transposon mutant pools derived from LB and minimal media were thawed on ice. To account for differences in cell density, 100 μl of the LB transposon mutant pool and 1 ml of the minimal medium transposon mutant pool were pelleted and resuspended in 1 ml TSB, and the cells densities for each transposon mutant pool were adjusted to OD600=1.0. These samples were diluted 1:200 in 12.5 ml of TSB in a 250 ml Erlenmeyer flask and incubated at 37°C for 24 hours. Cells from each culture were harvested by centrifugation, washed twice with DPBS and cell density was adjusted to OD600=1 with DPBS. The transposon mutant pools were combined in a 2:1 ratio of LB to minimal medium transposon mutant pool. Cell suspension (2 spots of 50 μl) was filtered and membranes were treated as described above, except in addition to viable counts, the remaining cells that were resuspended from the membrane (about 900 μl) were plated onto TSB in a Q-tray and incubated for 4-5 hours, which resulted in a thin film of growth. Cells were scraped, resuspended in 1 ml DPBS, centrifuged, and frozen at −80°C for Tn-seq library preparation. Two biological replicates of Tn-seq were performed.
Tn-seq library preparation, Illumina sequencing, and data analysis
Genomic DNA from samples desiccated for 0 or 6 days were extracted using the DNeasy Blood and Tissue Kit (Qiagen). Tn-seq libraries were prepared using the terminal deoxynucleotidyl transferase (TdT) method as previously described (24, 52), and sequencing was carried out on an Illumina Miseq platform. Total reads were ranged from approximately 3.8 to 7.7 million. Oligonucleotides used in this study are listed in Table S5B. Reads were normalized, mapped, and counted using custom Python scripts available at Github (https://github.com/elijweiss/Tn-seq). Reads were further normalized to gene length. To identify candidate genes important for desiccation resistance, reads from both biological replicates were averaged for samples desiccated for 0 or 6 days.
Construction of the ΔcsrA mutant
In-frame deletion of the csrA (ABUW_2750) gene was generated by overlap extension PCR as described (53). PCR primers are listed in Table S5B. PCR product was cloned into mobilizable suicide vector pEX2-TetRA and transformed into E. coli NEB 10-beta (New England Bio Labs). The sequence-verified deletion construct was transformed into E. coli S17-1, and further mobilized into A. baumannii strain AB5075 by conjugation on TY agar. Single recombinant conjugants were first selected on M9/succinate plate containing 20 μg/ml Tc, and Tc resistant colonies were further plated onto M9/succinate plate containing 5% sucrose. Sucrose resistant and Tc sensitive colonies were screened by colony PCR and sequencing to validate the expected chromosomal in-frame deletion of the csrA gene.
To complement the ΔcsrA mutant, the full length csrA gene including the 15 bp upstream that contains the putative ribosome binding site was PCR amplified and cloned into pMMB67EH-TetRA. The construct was transformed into E. coli NEB 10-beta (New England Bio Labs). The sequence-verified construct was transformed into E. coli S17-1, and further mobilized into the ΔcsrA mutant by conjugation on M9/succinate plate containing 20 μg/ml Tc. As a negative control, empty vector pMMB67EH-TetRA was used. The same procedure was used to clone ABUW_2433 and ABUW_2437 for complementation experiments.
Label-free protein quantification
Since the ΔcsrA mutant had severe growth defect on TY media, both wild type and ΔcsrA mutant were grown in M9/succinate. Two biological sample replicates were prepared for each strain. Cells from each culture were harvested at OD600=0.5 by centrifugation, washed twice with DPBS, and cells were stored in −80°C before further analysis. Cells were lysed in buffer containing 4% SDS, 100 mM Tris pH8.0, 10 mM DTT by heating at 95°C for 5 min. After cooling to room temperature, the lysates were sonicated with ultrasonication probe on ice to shear DNA. Total protein concentration was determined by the BCA assay (Thermo Pierce, Rockford, IL). 500 μg of each protein lysate was reduced and alkylated, diluted in 8 M urea solution, and the SDS was removed with a 3kD molecular weight cutoff filter. After buffer exchange, the protein lysates were digested with trypsin (Promega, Madison, WI) at 37°C overnight and the digested samples were desalted with 1cc C18 Sep-Pak solid phase extraction cartridges (Milford, MA, Waters). The eluted samples were vacuum dried and resuspended in 0.1% formic acid. Reverse phase nanoLC-MS analysis of the protein digests was carried out with a Thermo Easy-nanoLC coupled to a Thermo Q-Exactive Plus Orbitrap mass spectrometer. Triplicate top 20 data-dependent acquisition runs were acquired for each sample, and 1 μg of protein digest was loaded for each run. The peptides were separated by a 50 cm x 75 μm I.D C8 column (5 μm diameter, 100 Å pore size C8 MichromMagic beads) with a 90 min 10 to 30% B gradient (solvent A: 0.1% formic acid in water, solvent B: 0.1% formic acid in acetonitrile, flow rate 300 nl/min). The MS data acquisition parameters were set as follows: full MS scan resolution 70k, maximum ion injection time 100 mS, AGC target 106, scan range of 400 to 2000 m/z; MS/MS scan resolution 17.5 k, maximum ion injection time 100 mS, AGC target 54, isolation window 1.6 m/z, HCD NCE 35 scan range of 200 to 2000 m/z; loop count 20, intensity threshold 53, underfill ratio 1%, dynamic exclusion 10 sec. High resolution MS2 spectra were searched against a target-decoy proteome database of strain AB5075 (a total of 7678 sequences) downloaded from Uniprot (Oct17, 2017) using Comet (version 2015.02 rev. 1) (54) with following parameters: precursor peptide mass tolerance 20 ppm, allowing for −1, 0, +1, +2, or +3 13C offsets; fragment ion mass tolerance 0.02 Da; static modification, carbamidomethylation of cysteine (57.0215 Da); variable modification, methionine oxidation (15.9949 Da). The search results were further processed by PeptideProphet (55) for probability assignment to each peptide-spectrum match, and ProteinProphet (56) for protein inference and protein probability modeling. The output pepXML files from three technical replicates were grouped for subsequent spectral counting analysis using Abacus (57). The pepXML and protXML files for each sample, combined ProteinProphet file from all samples were parsed into Abacus for spectral counting of each protein. The following filters were applied for extracting spectral counts from MS/MS datasets: (1) the minimum PeptideProphet score the best peptide match of a protein must have maxIniProbTH=0.99; (2) The minimum PeptideProphet score a peptide must have to be even considered by Abacus, iniProbTH=0.50; (3) The minimum ProteinProphet score a protein group must have in the COMBINED file, minCombinedFilePw=0.90. Spectral counts for 1616 proteins were reported across four sample groups (two strains and two biological replicates) with estimated protein false discovery rate of 1.94%. The protein expression fold changes between wild type AB5075 and ΔcsrA mutant were computed from adjusted spectral counts output from Abacus.
Phenotypic characterization of the ΔcsrA mutant
M9/succinate was used in all experiments. For motility assays, an overnight culture (16 to 18 hours) was diluted to yield OD600=0.5, and 2 μl of sample was spotted onto the freshly prepared M9/succinate plate and incubated at 37°C for 24 h. For biofilm assays, an overnight culture was inoculated into 100 μl of M9/succinate in Costar vinyl 96 well “U” bottom plates (initial OD600=0.05), and the plates were sealed with Breath-Easy sealing membranes. After incubation at room temperature for 48 hours, culture was removed, the plate was rinsed with tap water twice, and 150 μl of 0.1% crystal violet solution was added to each well. After incubating at room temperature for 15 min, crystal violet solution was removed, the plate was rinsed with tap water 5 times, and the plate was dried at room temperature. For catalase assays, cells were harvested at OD600=0.5, supernatant was removed, and cells were resuspended in DPBS to yield 10 mg wet cell/100 μl DPBS. 100 μl of cell suspensions were placed in 13 x 100 mm borosilicate glass tubes. Then 100 μl of 1% Triton X-100 and 100 μ1 of 30% hydrogen peroxide were added, mixed thoroughly, and incubated for 15 min at room temperature (58).
FUNDING
This work was supported by the Functional Genomics Program, National Institute of Allergy and Infectious Diseases under Grant 1U19AI107775-01.
SUPPLEMENTAL MATERIAL
Table S1. Screening of desiccation sensitive mutant by Tn-seq.
Table S2. Mutants from the three-allele library tested for desiccation tolerance
Table S3. (A) Label-free protein quantification of wild type and the ΔcsrA mutant. (B) List of proteins up-regulated (ratio of ΔcsrA/WT ≥2.5 and ΔcsrA read count ≥7.5) in the ΔcsrA mutant compared to wild type. (C) List of proteins down-regulated (ratio of ΔcsrA/WT ≤0.4 and WT read count ≥7.5) in the ΔcsrA mutant compared to wild type.
Table S4. Validation of desiccation phenotype of transposon mutant derived from the ΔcsrA mutant proteomics analysis.
Table S5. Bacterial strains used in this study (A). Plasmids and primers used in this study (B).
ACKNOWLEDGMENTS
We thank Dr. Hemantha Don Kulasekara and Dr. Samuel Miller (University of Washington) for sharing the pEX2-TetRA and pMMB67EH-TetRA vectors. We thank Indranil Biswas for alerting us to the possibility that intrinsically disordered proteins could be involved in desiccation tolerance in A. baumannii.