Pathogenic Acinetobacter species have a functional type I secretion system and contact-dependent inhibition systems

Pathogenic Acinetobacter species have a functional type I secretion system and contact-dependent inhibition systems Received for publication, February 14, 2017, and in revised form, March 31, 2017 Published, Papers in Press, April 3, 2017, DOI 10.1074/jbc.M117.781575 Christian M. Harding ‡1,2 , Marina R. Pulido ‡§1,3 , Gisela Di Venanzio ‡ , Rachel L. Kinsella ‡¶4 , Andrew I. Webb 储 **, Nichollas E. Scott ‡‡5 , Jerónimo Pachón § , and Mario F. Feldman ‡6 From the ‡ Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110, the § Unit of Infectious Diseases, Microbiology, and Preventive Medicine and Biomedical Institute of Seville, University Hospital Virgen del Rocío/Consejo Superior de Investigaciones Científicas, University of Sevilla, 41004 Seville, Spain, the ¶ Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2R3, Canada, the 储 Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria 3052, Australia, the **Department of Medical Biology, University of Melbourne, Parkville, Victoria 3050, Australia, and the ‡‡ Department of Microbiology and Immunology, Doherty Institute, University of Melbourne, Melbourne, Victoria 3000, Australia Edited by Chris Whitfield Pathogenic Acinetobacter species, including Acinetobacter baumannii and Acinetobacter nosocomialis, are opportunistic human pathogens of increasing relevance worldwide. Although their mechanisms of drug resistance are well studied, the virulence factors that govern Acinetobacter pathogenesis are incompletely characterized. Here we define the complete secretome of A. nosocomialis strain M2 in minimal medium and demonstrate that pathogenic Acinetobacter species produce both a functional type I secretion system (T1SS) and a contact-dependent inhibition (CDI) system. Using bioinformatics, quantitative proteomics, and mutational analyses, we show that Acinetobacter uses its T1SS for exporting two putative T1SS effectors, an Repeatsin-Toxin (RTX)-serralysin-like toxin, and the biofilm-associ- ated protein (Bap). Moreover, we found that mutation of any component of the T1SS system abrogated type VI secretion activity under nutrient-limited conditions, indicating a previously unrecognized cross-talk between these two systems. We also demonstrate that the Acinetobacter T1SS is required for biofilm formation. Last, we show that both A. nosocomialis and A. baumannii produce functioning CDI systems that mediate growth inhibition of sister cells lacking the cognate immunity protein. The Acinetobacter CDI systems are widely distributed across pathogenic Acinetobacter species, with many A. baumannii isolates harboring two distinct CDI systems. Collectively, these data demonstrate the power of differential, quantitative proteomics approaches to study secreted proteins, define the role of previously uncharacterized protein export systems, and observe cross-talk between secretion systems in the pathobiology of medically relevant Acinetobacter species. Medically relevant Acinetobacter species are nosocomial pathogens accounting for ⬃2% of all healthcare-associated infections in the United States (1). More alarming has been the increasing rate of multidrug resistance (MDR) 7 phenotypes associated with Acinetobacter-induced infections; so much so that the CDC has listed Acinetobacter as a severe threat, and last-line therapeutic options like carbapenems, tigecycline, and colistin are now the recommended treatment options for the majority of Acinetobacter infections (2, 3). Furthermore, recent epidemiological data indicate that Acinetobacter spp. appear to be acquiring MDR phenotypes faster than almost all other Gram-negative bacteria over the last decade (4). For decades, the causative agent behind Acinetobacter infections was almost exclusively reported as Acinetobacter baumannii; however, Acinetobacter taxonomy has undergone major modifications over the last several years. Specifically, we now know that medically relevant Acinetobacter spp. predominately come from a single group, designated the Acinetobacter baumannii (Ab) group, given their close genetic relatedness and indistinguishability at the phenotypic level when examined in clinical laboratories. Members of the Ab group include A. baumannii,Acinetobacter nosocomialis,Acinetobacter pittii,Acinetobacter seifertii, and Acinetobacter dijkshoorniae, all of which act as opportunistic pathogens and acquire MDR phenotypes (5–7). Nevertheless, A. baumannii still remains the most comprehensively studied member of the Ab group, with A. nosocomialis a distant second. The authors declare that they have no conflicts of interest with the contents of this article. This article contains supplemental Figs. 1–3, Tables 1 and 2, and a Mass Spectrometry Table. The mass spectrometry proteomics data have been deposited to the Proteome- Xchange Consortium via the PRIDE partner repository with the dataset identifier PXD005881. 1 Both authors contributed equally to this work. 2 Funded as a W. M. Keck Postdoctoral Fellow during the course of this study. 3 Supported by the Subprograma Sara Borrell from the Instituto de Salud Carlos III, Subdirección General de Evaluación y Fomento de la Investigación, Ministerio de Economía y Competitividad of Spain (CD14/00014). 4 Funded by Natural Sciences and Engineering Research Council of Canada (NSERC). 5 Supported by a National Health and Medical Research Council of Australia (NHMRC) Overseas (Biomedical) Fellowship APP1037373, NHMRC Project Grant APP1100164, and the University of Melbourne Early Career Researcher Grant Scheme (Proposal 603107). 6 To whom correspondence should be addressed. Tel.: 314-747-4473; Fax: 314-362-1232; E-mail: [email protected]. 7 The abbreviations used are: MDR, multidrug resistance; Ab,Acinetobacter baumannii; T2SS, type II secretion system(s); T4P, type IV pili; CDI, contactdependent inhibition; ABC, ATP-binding cassette; Bap, biofilm-associated protein; RND, resistance-nodulation-cell division; ANOVA, analysis of variance; RTX, repeat-in-toxin. cros ARTICLE J. Biol. Chem. (2017) 292(22) 9075–9087 9075 © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Although our understanding of the mechanisms that mediate MDR phenotypes is quite comprehensive (8), those that mediate Acinetobacter pathogenesis are only beginning to be defined. The role of secretion systems in Acinetobacter virulence has been receiving more and more attention because of their importance in other clinically relevant pathogens. Multiple secretion systems, including a type II secretion system (T2SS), a type V secretion system (T5SS), and a T6SS, have all been characterized and attributed specific roles in the pathobiology of Acinetobacter (9–13). Both the T2SS and T5SS have clear roles in pathogenesis, as specific exported factors have been directly linked to virulence, including the type II effector CpaA and the type V autotransporter Ata (11, 14). The T6SS, not yet linked directly to eukaryotic pathology, is responsible for killing bacteria and commonly found to be active in clinical isolates extracted from polymicrobial infections, indicating that it may impart a fitness advantage in vivo (15). Furthermore, plasmid-borne conjugation systems similar to a T4SS have also been identified in A. baumannii (16), but detailed molecular analyses describing these systems are lacking. Lastly, type IV pili (T4P), bacterial grappling hooks evolutionarily related to the T2SS, have also been characterized in Acinetobacter for their role in natural transformation, motility, and biofilm formation (17, 18). Given the extensive genetic variation of Acinetobacter spp. and our lack of a thorough understanding of their molecular pathogenesis, we hypothesized that other unrecognized secreted factors may be contributing toward Acinetobacter fitness and virulence. To this end, we bioinformatically identified, in Ab group member genomes, a T1SS and contactdependent inhibition (CDI) systems. The T1SS of Acinetobacter is homologous to the prototypical TolC-HlyD-HlyB system from Escherichia coli for export of the hemolysin HlyA (19). This three-component secretion system requires an inner membrane ATP-binding cassette (ABC) transporter, a periplasmic adaptor protein (PAP), and an outer membrane protein channel (20). In E. coli, TolC acts as the outer membrane protein and forms a homotrimer that embeds into the outer membrane, extending far into the periplasmic space, where it can interact with the periplasmic adaptor protein HlyD (21). The periplasmic adaptor protein HlyD, anchored by a transmembrane domain within the inner membrane (22), not only interacts with the outer membrane protein TolC and the ABC transporter HlyB but can also interact with the T1SS substrate via an N-terminal cytoplasmic region (23). The ABC transporter HlyB, also embedded within the inner membrane, hydrolyzes ATP to provide the energy required for export of a specific T1SS substrate (22). Together, these proteins act to facilitate the secretion of unfolded effectors directly from the cytoplasm to the extracellular environment. CDI systems, first discovered in 2005 (24), constitute a tripartite mechanism for inhibiting the growth of neighboring bacteria independent from T6SS. Both CdiB and CdiA are considered to be T5SS, where the outer membrane pore-forming CdiB protein facilities the export of a CdiA toxin to the cell surface. CdiA toxins are polymorphic, usually containing multiple filamentous hemagglutinin domains in the N-terminal region and a C-terminal toxin domain. The third component, the CdiI protein, acts as an immunity protein preventing autoinhibition. Immunity proteins commonly contain domains associated with the SUKH superfamily (25). A fourth component, the toxin receptor, is also required for CDI killing; however, these are not intuitively identifiable and are not associated with cdiBAI loci. To date, only a handful of other CDI systems (Dickeya dadantii (26), Burkholderia (26), Enterobacter cloacae (27), Pseudomonas aeruginosa (28), and Neisseria meningitidis (29)) have been functionally characterized. Here, utilizing a quantitative, differential proteomics technique paired with bioinformatics, we report the complete secretome of A. nosocomialis strain M2 and simultaneously identify two previously unrecognized secretion systems across medically relevant Acinetobacter species. Results Identification of a T1SS-associated locus and its role in the secreted protein profile of A. nosocomialis strain M2 Previously, we demonstrated that A. nosocomialis strain M2 (formerly designated A. baumannii strain M2 (30)) has a functioning T2SS that secretes multiple effectors, including the metalloprotease CpaA (10). Furthermore, given that strain M2 also encodes for a functioning T6SS (12), we hypothesized that other secretion systems not yet characterized may also be contributing to the secreted protein profile. Using a BLASTp search, we identified homologs of the T1SS components tolC, hlyB, and hlyD from E. coli (Fig. 1A). To determine whether this putative system was contributing toward the secreted protein profile in strain M2, we constructed a tolC-hlyB-hlyD::kan mutant (T1SS mutant). Using one-dimensional SDS-PAGE analysis, we found that the T1SS mutant exhibited an altered secreted protein profile, as evidenced by the absence of highmolecular-weight proteins at 70, 130, and 250 kDa compared with the parent (Fig. 1B). Importantly, the complemented strain displayed a secreted protein profile nearly identical to the parental strain, indicating that the absence of the tolC-hlyB- hlyD gene cluster was responsible for the observed phenotype (Fig. 1B). Next, we sought to further characterize these differences and simultaneously define the complete secretome of strain M2 in minimal medium. The complete secretome of A. nosocomialis strain M2 reveals T1SS-, T2SS-, T6SS-, and CDI-secreted candidates To define the complete secretome and simultaneously identify differentially secreted proteins between strain M2 and the T1SS mutant, we utilized a quantitative proteomics approach. Briefly, we enriched for secreted proteins from each strain using bacteria grown to mid-log in M9 minimal medium supplemented with 0.2% casamino acids. This method eliminated the use of rich medium containing high concentrations of yeast proteins as well as limited cellular lysis contaminants found in stationary-phase bacterial cultures. Using dimethyl labeling of enzymatically digested peptides obtained from the secreted protein fractions of each strain, we were then able to compare the relative quantities of all secreted proteins in pairwise fashions. Four biological samples were prepared for each strain and processed as described under “Experimental Procedures” prior Pathogenic Acinetobacter species have a T1SS and CDI system 9076 J. Biol. Chem. (2017) 292(22) 9075–9087 to LC/MS analysis. Isotopically labeled samples were then pooled, and the four biological dimethylation triplex experiments were analyzed by LC/MS. A sample of each replicate for each strain was run on an SDS-PAGE gel, silver-stained, and found to have similar loadings and reflected the overall secretion trend observed in Fig. 1 (supplemental Fig. 1). First we compiled a list of the top secreted proteins from the wild-type M2. Our list was ranked based on proteins with the highest number of unique identified peptides (arbitrarily cut off at 20 for Fig. 1C). The complete list of proteins can be found in the supplemental Mass Spectrometry Table. We identified components of the T6SS and the T2SS, which was expected given previous characterizations of these systems in pathogenic Acinetobacter spp. Furthermore, we identified proteins putativelyassociatedwiththeT1SSandaCDIsystem,bothofwhich are discussed in detail below. RTX and Bap are secreted via a T1SS Using the Log 2 ratio of high-confidence proteins (defined as proteins with ⱖ13 unique identified peptides) observed across replicates, we were able to identify multiple proteins that were significantly and differentially secreted by the wild type compared with the T1SS mutant (Fig. 2A). As expected, the top differentially secreted protein between the parent and the T1SS mutant was a predicted T1SS protein based on the presence of the type I secretion C-terminal target domain (VC_A0849) in the C terminus of the M215_09430 open reading frame. The M215_09430 locus encodes for a protein containing multiple peptidase M10 serralysin C-terminal domains, a Ca 2⫹ -binding protein RTX toxin-related domain, and the type I secretion C-terminal target domain (VC_A0849 subclass). For simplicity, we will refer to M215_09430 as the RTX protein. We also identified a second protein containing a type I secretion C-terminal Figure 1. Acinetobacter nosocomialis strain M2 encodes for a functional T1SS. A, genetic arrangement and locus identifiers for the T1SS locus in strain M2. Boxes below each gene contain the relevant protein family domain information associated with the respective amino acid sequence for TolC, HlyB, and HlyD, as determined by BLASTp. B, Coomassie-stained, one-dimensional SDS-PAGE analysis of the secreted protein profiles from the wild-type M2, the T1SS mutant (tolC-hlyD::kan), and the complemented T1SS mutant. Secreted proteins were enriched from bacteria grown overnight in rich medium. C, the most abundant high-confidence secreted proteins from strain M2 grown to mid-log in minimal medium. High-confidence proteins were defined as proteins with ⱖ20 unique identified peptides across replicates. The export column details the export/secretion mechanism for the each protein. The asterisk beside T1SS denotes putative secretion by the TolC-HlyB-HlyD system, as determined by the presence of the type I secretion C-terminal target domain (VC_A0848) for M215_09430 and M215_01695 and the absence of these proteins in our differential proteomics analysis comparing secreted proteins from the wild type and the T1SS mutant, described in detail in Fig. 2. Pathogenic Acinetobacter species have a T1SS and CDI system J. Biol. Chem. (2017) 292(22) 9075–9087 9077 Pathogenic Acinetobacter species have a T1SS and CDI system 9078 J. Biol. Chem. (2017) 292(22) 9075–9087 target domain, M215_01695, which was significantly and differentially secreted between the wild type and T1SS mutant. The M215_1695 locus encodes for a large protein predicted to containmultiplegroup3bacterial Ig-likedomains(pfam13754) and a type I secretion C-terminal target domain (VC_A0848). Given its homology to biofilm-associated proteins (Baps) in A. baumannii, this protein was designated the Bap ortholog in A. nosocomialis strain M2. The second most differentially secreted protein between the wild type and the T1SS mutant was M215_02910 (Fig. 2, Aand B), a protein predicted to contain multiple group 3 bacterial Ig-like domains (pfam13754) similar to Bap; however, the protein annotation for M215_02910 lacks the type I secretion C-terminal target domain commonly found in T1SS proteins. Recent molecular, in silico, and PCR analyses of bap genes from A. baumannii demonstrate that the bap repetitive elements are prone to alignment errors in genome sequences, resulting in premature truncations and chromosomal rearrangements (31– 33). Together, this may indicate that M215_02910 is part of the same open reading frame as Bap. Conversely, it is also possible thatthis open reading frame has been incorrectly alignedandis, in fact, part of the RTX protein, given its similarities to large repetitive RTX proteins (34). Regardless, the presence of peptides from this protein in the secreted fraction of both the wild type and complemented strain strongly correlate with its secretion with the T1SS. Mutation of the T1SS reveals cross-talk between other secretion systems Analysis of our proteomics results indicated that proteins without the type I C-terminal targeting domain were also differentiallysecreted between the wildtypeand the T1SS mutant. Specifically, multiple T6SS-associated proteins, including three VgrG orthologs and Hcp, were all found at significantly lower levels in the T1SS mutant. Although not nearly as significant, multiple known T2SS-associated proteins, including CpaA, LipH, a rhombosortase, and a rhombotarget, were also found in lower quantities in the T1SS mutant compared with the wild type. Last, a predicted CDI-associated protein was also identified as significantly reduced in the T1SS mutant (discussed in detail below). The full list of differentially secreted proteins can befound inthe supplementalMassSpectrometry Table.For the majority of instances, including the predicted T1SS-associated proteins described above, the dimethylation ratios comparing the secreted proteins from the complemented strain with the parent strain were quite similar, again indicating that the absence of the tolC-hlyB-hlyD genes was responsible for the differential secretion profile in the mutant. Deletion of the T1SS systems represses T6SS activity in minimal medium only It has been shown previously that deletion of tolC can have pleiotropic effects on membrane integrity and cellular physiology in E. coli (35). Therefore, we initially hypothesized that the observed differences between the secreted proteins of the parent and T1SS mutant were due to membrane stress associated with the absence of the TolC protein. To gain insight into the molecular mechanism behind the diminished protein profile of the T1SS mutant and more intricately probe the role of tolC, hlyB, and hlyD in the observed phenotypes, we constructed in-frame unmarked mutations in each gene as well as the entire locus. First we validated the quantitative proteomics results by Western blotting across the entire mutant panel in minimal Figure 2. Quantitative proteomics analysis comparing secreted proteins from strain M2, the T1SS mutant, and the complemented strain demonstrates global changes to the secretome. A, heatmap of quantitative proteomics results of high-confidence proteins (defined as proteins with ⱖ13 unique identified peptides). The Log 2 ratio of high-confidence proteins observed across replicates demonstrates the loss of multiple proteins in response to disruption of T1SS (low ⌬tolC-hlyD/WT demethylation ratios). Upon restoration of T1SS in the form of the complemented strain, secreted protein profiles return to near wild-type levels (complement/WT demethylation ratios of ⬃0). B, scatterplot of quantitative proteomics results showing the ⫺log 10 (pvalues) compared with the average ⌬tolC-hlyD/WT ratios of alterations observed within the secretome of ⌬tolC-hlyD biological replicates compared with WT replicates. Statistically significant changes (p⬍0.05) are highlighted in red. Figure 3. Mutation of any component of the T1SS abrogates T6SS activity, as determined by Hcp secretion in minimal medium only. Aand B, Western blot analysis probing for Hcp expression and secretion from the wild type and T1SS mutant panel and complemented strains grown in minimal medium (A)or rich medium (B). Hcp expression in whole cells from strains grown in either minimal or rich medium was equal. However, Hcp secretion was found to be suppressed in strains with mutations in any component of the T1SS in minimal medium. Importantly, complemented strains had Hcp secretion levels approximately equivalent to the wild type, with the exception of the hlyB complement, which had an intermediate phenotype. As a loading and lysis control, RNA polymerase expression was probed for across all conditions. C, whole cells; S, secreted proteins. Pathogenic Acinetobacter species have a T1SS and CDI system J. Biol. Chem. (2017) 292(22) 9075–9087 9079 medium. As expected, Hcp expression was found to be similar across all strains tested; however, Hcp secretion was not detected in any of the mutants analyzed when secreted proteins were collected from strains grown in minimal medium (Fig. 3A). Interestingly, when the mutant panel was tested for Hcp secretion from strains grown in rich medium, no differences were observed in Hcp expression or secretion (Fig. 3B). Next we sought to determine whether the cross-talk between the T1SS system and the T6SS was specific or whether disrupting other secretions systems would also abrogate T6SS activity. To this end, we probed for Hcp expression and secretion in a pilD mutant and complemented strain. PilD is the pre-pilin peptidase required for processing of both pilins and pseudopilins for the T4P system and T2SS, respectively. Thus, the absence of pilD results in a T4P- and T2SS-negative phenotype (10). The pilD mutant displayed no differences in Hcp expression or secretion compared with the parental strain and the complemented strain. Furthermore, this phenotype held true for both rich and minimal medium (Fig. 4, Aand B). The T1SS system is required for biofilm formation Given that Bap was one of the most differentially secreted proteins between the wild type and the T1SS full locus mutant, we hypothesized that our unmarked individual T1SS mutants wouldhaveimpaired abilityto formbiofilms. Usinga microtiter plate biofilm assay, we observed that mutants lacking any or all components of the T1SS have severely reduced ability to form biofilms, as determined by crystal violet staining both 3 and 6 h after inoculation (Fig. 5, Aand B, respectively). Importantly, we performed biofilm formation assays in rich medium to avoid confounding results from altered secretion of non-T1SS components, like Hcp secretion, as shown in Fig. 3. Nevertheless, theattenuatedbiofilmphenotypewasobserved inboth richand minimal medium (data not shown). The biofilm defect was not due to alterations in Csu pili or T4P expression and surface presentation either. Specifically, the major pilin subunits PilA of the T4P and CsuA/B of Csu pili were equally expressed in whole cells and on the surface of all mutants and complemented strains (supplemental Fig. 2). Last, the biofilm phenotype was not due to differences in the second most abundantly secreted protein, M215_12375 (CdiA), as a cdi locus mutant had the same level of biofilm as the parent strain (supplemental Fig. 3). T1SS mutants appear to have attenuated virulence phenotypes in a Galleria mellonella infection model The greater wax moth, G. mellonella, is a commonly used model system for measuring Acinetobacter virulence and correlates well with in vivo virulence in mammalian systems (36). To assess the role of the T1SS in virulence, we individually infected larvae with the wild type, a T1SS mutant, or its respective complemented strain and monitored larva viability over 24 h. Larvae were injected with 10 ␮ l of a bacterial suspension determined previously to be the LD 50 for the wild-type M2 (10). Although not statistically significant, a clear virulence attenuation trend was observed in all T1SS mutants compared with the parental or complemented strains, indicating that the T1SS contributes to the pathogenicity of medically relevant Acinetobacter spp. (Fig. 6, A–D). Although the T1SS mutant has an altered T6SS phenotype in minimal medium only, we have demonstrated previously that T6SS mutants are equally as virulent as wild-type A. baumannii (13). Pathogenic Acinetobacter spp. utilize a contact-dependent inhibition system for bacterial killing Two of the most abundantly secreted proteins, based on observed peptides within our datasets, were the RTX protein and M215_12375. Both proteins show profound alterations in their secretion between the T1SS mutant and the wild-type strain. As mentioned above, the RTX protein is an RTX serralysin-like protein, a common class of toxins secreted by T1SS (37),whereastheM215_12375 locusencodes fora polymorphic toxin similar to those found in CDI systems (Fig. 7A). Given its homology to CDI systems and the data presented below, we havedesignated thisgenecluster thecdiBAI M2 system(Fig. 7A). Figure 4. Mutation of both the T2SS and T4P system does not abrogate T6SS. Aand B, Western blot analysis probing for Hcp expression and secretion from wild-type M2, a pilD mutant, and the pilD complemented strain grown in minimal medium (A) or rich medium (B). Both Hcp expression and secretion were equivalent across strains grown in minimal medium or rich medium. As a loading and lysis control, RNA polymerase (RNAP) expression was probed for across all conditions. Pathogenic Acinetobacter species have a T1SS and CDI system 9080 J. Biol. Chem. (2017) 292(22) 9075–9087 To determine whether the cdiBAI M2 system was active in strain M2, the entire locus was replaced with a kanamycin cassette, generating the M2⌬cdi mutant. Furthermore, we generated a variant of the cdi mutant expressing just the cdiI immunity gene in trans. Next we performed inhibition assays in pairwise fashions. As seen in Fig. 7B, M2 is able to inhibit the growth of the cdi mutant by ⬃1 log compared with co-incuba- tions between the cdi mutant and the cdi mutant expressing just the immunity protein. Furthermore, M2 is not able to inhibit the growth of the cdi mutant expressing just the immunity protein, indicating that CdiI is indeed the immunity protein for this cdi system. Last, CDI killing was found to be T6SS- independent,as an hcp mutant co-incubated with a double hcp/ cdi mutant had an indistinguishable phenotype from wild-type M2 co-incubated with the cdi mutant (Fig. 7B). CDI systems are conserved in medically relevant Acinetobacter To expand our findings to the majority of medically relevant Acinetobacter spp.,webioinformaticallyidentifiedCDIsystems in A. baumannii 19606 and 1225, two clinical isolates representing a type strain as well as newer, more relevant clinical isolate, respectively. Interestingly, both A. baumannii 19606 and 1225 encode for two predicted cdi loci, a trait common to P. aeruginosa (28). CDI loci from 19606 and 1225 were similar in gene architecture to A. nosocomialis strain M2 with a putative transporter protein, the polymorphic toxin, and an immunity protein. For both strains, each cdi locus was individually mutated, generating cdi_1 and cdi_2 mutants for both 19606 and1225.Subsequently,thecdi_1andcdi_2mutantswereindi- vidually co-incubated with their respective isogenic parent strains for determination of growth inhibition. Similarly to the strain M2 co-incubations, 19606 and 1225 mutants lacking their respective cdi systems had their growth inhibited by ⬃1 log compared with co-incubations between the wild-type strain and a cdi mutant complemented with the respective immunity protein (Fig. 7, Cand D). In each case, the difference between the parent strain and a cdi mutant was statistically significant, whereas co-incubations between a parent strain and a cdi mutant expressing the immunity protein closely reflected the growth of the cdi mutant in the absence of an inhibitor strain (Fig. 7, Cand D,last columns). Discussion Pathogenic Acinetobacter spp., including A. baumannii and A. nosocomialis, are opportunistic pathogens with an unmatched ability to acquire MDR phenotypes. Although the mechanisms behind drug resistance are well documented, the full slate of virulence factors that mediate virulence are not yet defined. In this work, we applied quantitative proteomics techniques to identify previously uncharacterized, secreted Acinetobacter proteins. This powerful approach, coupled with previous secretion system characterizations, facilitated the assignment of the complete secretome of A. nosocomialis strain M2 in minimal medium. Importantly analysis of the top secreted candidates revealed the presence of a functional T1SS and identified two putative T1SS effectors. Last, we found functional CDI systems in both A. nosocomialis strain M2 and two A. baumannii clinical isolates, 19606 and 1225. Bacterial interactions with the extracellular environment are commonly mediated through secreted proteins; however, the full arsenal of Acinetobacter secretion systems compared with other classical pathogens like P. aeruginosa is significantly undercharacterized. Nevertheless, a broad collection of publically available sequenced Acinetobacter genomes has allowed the in silico identification of previously characterized secretion systems important for pathogenesis in other bacteria. Using this approach, we were able to identify a T1SS locus in A. nosocomialis strain M2, homologous to the E. coli prototype T1SS containing tolC,hlyB, and hlyD genes (19). Unlike E. coli, however, the tolC,hlyB, and hlyD genes of strain M2 are in a threegene cluster and are most likely in an operon, given that the open reading frame for hlyB overlaps with both tolC and hlyD. Furthermore, this gene arrangement is commonly found across medically relevant Acinetobacter spp., including A. baumannii, with 88% identity conserved to the nucleotide level for commonly used laboratory strains like A. baumannii ATCC 17978, AB5075, AYE, ACICU, and AB307-0294. Furthermore, homo- Figure 5. The Acinetobacter T1SS is required for biofilm formation. Aand B, biofilm formation was assessed by crystal violet staining. Plates were statically incubated at 37 °C for3h(A)or6h(B). After the designated time, an aliquot of the bacterial culture was removed for A 600 readings. Wells were subsequently washed and stained for 10 min with 0.1% crystal violet. Excess crystal violet was removed by washing and solubilized by addition of 30% acetic acid. Biofilm formation was assessed by comparing the absorbance ratio at 550 nm for crystal violet and 600 nm for bacteria. As seen in both panels, T1SS mutants had significantly reduced ability to form biofilms; however, complemented strains had biofilm phenotypes similar to wild-type M2. For statistical analyses, one-way ANOVA with Tukey’s correction for multiple comparisons was performed. *, p⫽0.01; **, p⫽0.001; ***, p⫽0.0001. Pathogenic Acinetobacter species have a T1SS and CDI system J. Biol. Chem. (2017) 292(22) 9075–9087 9081 logs of both bap and the RTX protein are also found in these strains, highlighting their probable secretion by the T1SS. The Acinetobacter T1SS is related to other ABC transporters, like the resistance-nodulation-cell division (RND) efflux pumps. Many RND pumps, such as AdeABC, AdeFGH, and AdeIJK, have been extensively characterized in A. baumannii; however, their roles in the pathobiology of Acinetobacter have been linked directly with the efflux of small molecules, partic- Figure 6. T1SS have an attenuated virulence phenotype in the G. mellonella infection model. A–D, groups of G. mellonella larvae were injected with 10 ␮ l of either the parent strain, the tolC mutant (A), the hlyB mutant (B), the hlyD mutant (C), the full locus mutant (D), or the respective complemented strains at an inoculum previously determined to be the equivalent of the LD 50 for wild-type M2. Larvae were checked for viability as determined by melanin accumulation and motility. Although not statistically significant as determined by Mantel-Cox comparison of survival curves, a clear attenuation trend was observed for all mutants tested. Figure 7. Pathogenic Acinetobacter species encode for functional CDI systems. A, genetic arrangement and locus identifiers for the CDI locus in strain M2. Boxes below each gene contain the relevant protein family domain information associated with the respective amino acid sequence for CdiB, CdiA, and CdiI, as determined by BLASTp. B, growth inhibition assays for strain M2 cdi mutants. Strain M2 is able to inhibit the growth of the cdi mutant at a statistically significant level; however, co-incubation between the cdi mutant and the cdi mutant complemented with the immunity protein alone, or between the wild type and the cdi mutant complementedwiththeimmunity protein,did notshow statisticallysignificant differences.CDI-mediated growthinhibition wasindependent ofthe T6SS,as shownin thelast column.CandD,growthinhibitionassaysinA. baumannii19606(C)andthe A.baumannii1225 (D)mutantpanel.Both19606and1225encodefortwocdiloci,andboth are able to inhibit the growth of mutants lacking the cognate immunity proteins. For statistical analyses, one-way ANOVA with Tukey’s correction for multiple comparisons was performed for Band C, whereas a non-parametric Kruskal-Wallis test with Dunn’s correction for multiple comparisons was utilized forD. The CDI loci for 19606 can be found with the locus tags F911_RS14340 to F911_RS14350 for cdi_1 and F911_RS17415 to F911_RS17425 for cdi_2. Pathogenic Acinetobacter species have a T1SS and CDI system 9082 J. Biol. Chem. (2017) 292(22) 9075–9087 ularly antibiotics, and not protein substrates (38–40). However, Yoon et al. (41) demonstrated that overexpression of the RND pumps AdeABC and AdeIJK alters the composition of the Acinetobacter outer membrane, as evidenced by the repression of Csu pili, OmpA, and some metabolism-associated proteins, demonstrating the importance of ABC transporters in outer membrane homeostasis. In a related fashion, we observed that mutations in the T1SS altered the secretion profile compared with wild-type M2. Specifically, our analysis identified that the T6SS is affected by alterations to the T1SS, but only under nutrient limitation. Furthermore, this abrogation is not due to membrane stress associated with mutations in tolC, as mutants lacking the periplasmic adaptor protein HlyD or the ATPase HlyB also displayed thesame phenotype.Evenmore intriguing isthat this reduction in T6SS activity is specific for mutations in the T1SS, as mutations that alter both T2SS and T4P functionality had not effect on Hcp secretion. The fact that T6SS is only abrogated under nutrient limitation in T1SS mutants suggests a regulatory cross-talk that favors the functional activity of these two systems simultaneously, which has yet to be fully explained. These results have parallels with cross-talk between secretion systems in other bacteria. Specifically, others have shown that the T2SS and T6SS of P. aeruginosa are co-regulated by quorum sensing (42, 43), and transcription factors co-regulate the T3SS and T6SS of Burkholderia cenocepacia (44). The regulatory mechanism behind the Acinetobacter Hcp secretion repression in T1SS mutants has yet to be determined but is not dependent on the previously described plasmid regulation system found in A. baumannii 17978 (15), given that M2 does not carry a homologous plasmid. Nevertheless, the levels of Hcp expression in whole cells were equivalent across strains. Further, hcp is predicted to be co-transcribed with the complete T6SS biogenesis machinery in a single operon. Based on these observations, we hypothesize that the down-regulation of T6SS in the T1SS- deficient strains takes place at the posttranscriptional level. Severalother Acinetobacter isolates also express Hcpbutdo not constitutively secrete Hcp (13). Additional work is needed to understand T6SS regulation in Acinetobacter and its connections with other cellular processes. Two of the most abundantly secreted proteins, the RTX protein and Bap, relied on the T1SS for export, as evidenced by their relative absence in the T1SS mutant and the presence of a type I C-terminal target domain (VC_A0849) in both proteins. Little is known about the role of RTX domain-containing proteins in the pathobiology of Acinetobacter; however, De Gregorio et al. (33) have bioinformatically found that some Acinetobacter RTX domain-containing proteins also have bacterial Ig-like domains found in Bap orthologs. These proteins, like ABAYE0821 from A. baumannii AYE, also contain the same peptidase M10 serralysin C-terminal domain found in the M2 RTX protein. Interestingly, all or some of these domains are alsocommonly found in other well studied large repetitive RTX proteins, like LapA from Pseudomonas fluorescens and Pseudomonas putida as well as SiiE from Salmonella enterica (34). Asshownin ourproteomics data,the secondmost differentially secreted protein between the wild type and the T1SS mutant was the M215_02910 protein, which also contained multiple bacterial Ig-like domains but not a type I C-terminal target domain. It is tempting to speculate that these two proteins (RTX and M215_02910) are actually a single protein constituting a new member of the large repetitive RTX protein family; however, multiple unsuccessful attempts were made to clone and resequence the RTX gene. We hypothesize that, because of the presence of extensive repetitive sequences in these genes, the original genome sequence may be incorrect. Resequencing the genome of this strain will be necessary to properly assemble these genes. Although little is known about Acinetobacter large repetitive RTX proteins, Bap orthologs have been studied in detail for their role in Acinetobacter virulence and biofilm formation (45–47). From a molecular standpoint, Bap has been shown to be localized on the surface of A. baumannii (45) and, as we have shown, was readily identified in the culture supernatant. We have also shown that Bap is likely secreted by the T1SS. These results are congruent with those found for secretion of LapA, whereby a T1SS-like system, designated LapEBC, is responsible for its secretion (48). Nevertheless, molecular tagging experiments are required to definitively link Acinetobacter Bap secretion with the T1SS. From a phenotypic perspective, though, it is likely that the attenuated biofilm formation was due to the absence of Bap, given its previously characterized role in higherorder biofilm structure and maintenance (33). During the course of our characterization of the T1SS, we identified a CdiA M2 polymorphic toxin. CDI systems, composed of an outer membrane pore-forming protein, the polymorphic toxin, and an immunity protein, deliver toxic effectors to neighboring bacteria to inhibit growth. CdiA toxins are usually large, repetitive proteins with multiple N-terminal hemagglutinin repeats and a polymorphic C-terminal toxin domain (49). To date, CDI systems in Acinetobacter have been mentioned minimally in published reports, essentially just in bioinformatics comparisons of other previously well characterized systems (50–52), and none have shown any functional demonstration of activity. Although we demonstrated that CDI systems are functional, we did not identify the toxin receptor or the proposed mechanism of killing. However, the CdiA M2 toxin contains domains associated with DNase/tRNase activity, indicating that it may function as a cytoplasmic toxin degrading nucleic acids. Aside from the assigned toxic activities, novel signaling roles have been assigned to CDI systems, including signalingtocoordinatecooperationbetween bacteria that carry the same cdi alleles (53). Given that CDI systems appear widespread in Acinetobacter and that Acinetobacter is commonly associated with polymicrobial infections, these systems may also function as signaling molecules to coordinate killing activity in vivo. Recently, a new contact-dependent killing system, designated the Cdz system, was discovered in Caulobacter crescentus and was also bioinformatically identified in A. baumannii (54). These systems share similarities with CDI systems but rely on a T1SS for export of the surface-associated toxins. Further, the authors were able to identify the putative toxins in A. baumannii. Unfortunately, a BLASTp analysis revealed that there are no orthologs in A. nosocomialis M2 for these putative A. baumannii Cdz toxins. Nevertheless, it is likely that, in other Acinetobacter strains encoding for Cdz toxins, a T1SS is responsible for their secretion. Pathogenic Acinetobacter species have a T1SS and CDI system J. Biol. Chem. (2017) 292(22) 9075–9087 9083