Abstract
The Enterobacter cloacae complex (ECC) consists of closely-related, but genetically distinct bacteria commonly associated with the human microbiota. ECC have been increasingly isolated from healthcare-associated infections, demonstrating that these Enterobacteriaceae are emerging nosocomial pathogens. ECC strains can rapidly acquire multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the highly conserved lipid A component of the Gram-negative outer membrane to lyse the bacterial cell. Many Gram-negative Enterobacteriaceae fortify their outer membrane with cationic amine-containing moieties to protect from CAMP-inflicted lysis. The PmrAB two-component system (TCS) transcriptionally activates 4-amino-4-deoxy-L-arabinose (L-Ara4N) biosynthesis to result in amine moiety addition to lipid A in many Enterobacteriaceae such as E. coli and Salmonella. In contrast, PmrAB in E. cloacae is dispensable for CAMP resistance. Instead, fitness against CAMPs presents as heteroresistance, or a subpopulation of cells that exhibit clinically significant increases in resistance levels compared to the majority population. We demonstrate that E. cloacae lipid A is modified with L-Ara4N to induce CAMP heteroresistance and that the regulatory mechanism is independent of the PmrABEcl TCS. We show that the response regulator, PhoPEcl, directly binds to the arnBEcl promoter to induce expression of L-Ara4N biosynthesis and PmrAB-independent addition to the lipid A disaccharolipid. Therefore, we have identified a mechanism of ECC colistin heteroresistance that directly involves the PhoPQ system.
Importance Members of the Enterobacter cloacae complex (ECC) are Gram-negative nosocomial pathogens that have emerged within healthcare facilities around the world. ECC infections are associated with immunocompromised patients and infections are often life threatening. The cationic antimicrobial peptide, colistin (polymyxin E), is a last-line treatment option to combat Gram-negative multidrug resistant infections. However, many ECC intrinsically encode a colistin heteroresistance mechanism. Our analysis to characterize colistin heteroresistance in E. cloacae revealed that 4-amino-4-deoxy-L-arabinose is conjugated to the lipid A disaccharolipid to protect from colistin-mediated lysis. Additionally, this mechanism is directly regulated by the PhoPQEcl two-component system. Elucidation of outer membrane antimicrobial resistance modifications and their regulatory pathways in E. cloacae isolates will advance our understanding of CAMP heteroresistance.
Introduction
Gram-negative bacteria assemble a highly conserved outer membrane (OM) barrier, which restricts diffusion of toxins such as antibiotics into the cell. Glycerophospholipids comprise the periplasmic monolayer of the asymmetric lipid bilayer, while the surface-exposed monolayer is enriched with lipopolysaccharide (LPS). The LPS glycolipid is organized into three domains; the O-antigen carbohydrate repeat, core oligosaccharide, and the membrane anchor, lipid A (1). The lipid A domain is the bioactive portion of LPS and robustly activates the human Toll-like receptor 4 (TLR-4) and myeloid differentiation factor 2 (MD-2) immune complex to induce an immune response (1–4). Gram-negative pathogens encode highly conserved regulatory mechanisms to promote survival by responding to immune and environmental signals (5–7). Specific signaling pathways regulate lipid A modifications to alter TLR-4/MD-2 recognition and to fortify the OM against immune effectors and antimicrobials, which promotes survival in the host (8).
Lipid A modification enzymes are transcriptionally regulated by two-component systems (TCS) (9, 10). A prototypical TCS consists of an inner membrane sensor histidine kinase (HK) that senses a specific signal and a cognate cytoplasmic response regulator (RR), which alters expression of target genes. Signal recognition induces phosphotransfer and activation of the cognate RR, which typically results in DNA binding to alter gene expression (11). Most HKs encode a phosphatase domain that dephosphorylates the RR when the activating signal is depleted (6, 12). These highly conserved signaling systems enable bacteria to tightly regulate expression of target genes.
The PmrAB and PhoPQ TCSs are well-studied phosphorelay signaling systems that regulate lipid A modifications in response to specific environmental signals (13–15). PmrAB and PhoPQ are highly conserved among pathogenic Enterobacteriaceae, including Citrobacter, Escherichia, Klebsiella, Salmonella, and Shigella (5). PmrAB responds to high Fe3+ concentrations, cationic antimicrobial peptides (CAMPs), and slightly acidic pH to directly activate eptA (also known as pmrC) and arn operon expression (16–18), which encode phosphoethanolamine (pEtN) and 4-amino-4-deoxy-L-arabinose (L-Ara4N) transferases, respectively (19–22). Both enzymes transfer the respective amine-containing chemical moiety onto lipid A at the inner membrane, prior to LPS surface transport (19, 22). Cationic amine addition to the lipid A domain of LPS neutralizes the surface charge to protect the cell from CAMP-mediated lysis (19, 21).
PhoPQ is activated in response to depletion of divalent cations such as Mg2+ and Ca2+ and the presence of CAMPs (13, 15, 23). PhoPQ phosphotransfer directly activates transcription of genes encoding PagL (only in Salmonella (8)) and PagP, which add or remove acyl chains from lipid A, respectively (13, 24–26). While the PmrAB and PhoPQ TCSs each regulate distinct subsets of genes, the independent signaling pathways also converge through crosstalk (27, 28); PmrAB-dependent gene expression is also indirectly regulated by the PhoPQ TCS through the intermediate protein, PmrD. PmrD binds phospho-PmrA to prevent PmrB-mediated dephosphorylation (27, 29–31). Constitutive PmrA-dependent gene expression increases pEtN and L-Ara4N lipid A modifications.
The Enterobacter cloacae complex (ECC) is composed of thirteen closely-related Gram-negative bacterial clusters (designated C-I to C-XIII) (32). ECC are typically associated with the host microbiota, but many clusters cause hospital-acquired infections, especially in immunocompromised patients (33). Infections manifest in a wide range of host tissues with symptoms including skin, respiratory tract, urinary tract, wound and blood infections (34). ECC infections have increasingly emerged in nosocomial settings and are problematic because they encode multidrug resistance (MDR) mechanisms, which limits treatment options (33, 35–37). Alternative last-line therapeutics used to treat MDR Gram-negative infections include the CAMP, colistin (polymyxin E), which binds the lipid A portion of LPS to perturb the outer membrane and lyse the bacterial cell. Despite the success of colistin treatment as a last-line therapeutic to combat Gram-negative infections (38, 39), many ECC clusters demonstrated heteroresistance, where a subset of the clonal population is colistin resistant (35, 40–42). We do not fully understand the underlying molecular mechanism(s) that regulate colistin heteroresistance in ECC; further characterization will advance our understanding of antimicrobial resistance and could help improve treatment strategies.
Previous reports showed that colistin heteroresistance naturally occurs within clonal ECC clusters, including E. cloacae (cluster XI) (35). Moreover, colistin heteroresistance in E. cloacae was induced by innate immune defenses within a murine infection model to lead to treatment failure (40). Transcriptional analysis of susceptible and resistant populations suggested that pEtN and L-Ara4N lipid A modifications contribute to heteroresistance (40) and PhoPQ contributed to regulation (35, 40), as described in other Enterobacteriaceae (5). However, it was not established that the lipid A modifications actually occur, nor has PhoPQ-dependent, PmrAB-independent regulation of colistin heteroresistance been fully described in E. cloacae or other ECC isolates.
Herein, we demonstrate that E. cloacae colistin heteroresistance is directly regulated by PhoPQEcl to induce L-Ara4N modification of lipid A. In contrast, many other Enterobacteriaceae directly regulate L-Ara4N modification of lipid A via the PmrAB TCS. The PhoPEcl response regulator directly binds to the promoter region of arnBEcl, which is the first gene of a seven-gene operon (arnBCADTEFEcl). In contrast, PhoPEcl does not bind the arnB promoter region in E. coli. Transcriptomics analysis supports a model of PhoPQ-dependent, PmrAB-independent arnEcl regulation. Furthermore, L-Ara4N modification of lipid A increased in response to growth in limiting Mg2+, which induced colistin resistance in a PhoPQEcl-dependent manner.
Results
Colistin heteroresistance in E. cloacae is regulated by PhoPQEcl, but not PmrABEcl
To elucidate the underlying mechanisms that regulate colistin heteroresistance in ECC, we analyzed a collection of E. cloacae subsp. cloacae strain ATCC 13047 genetic mutants by calculating the colony forming units (CFUs) during exponential growth in the absence and presence of colistin (Fig 1A). While wild type and all mutant E. cloacae strains grew in standard growth media, ∆phoPQEcl was not viable when 10 μg/ml of colistin was added to the media. Clinical resistance to colistin is defined as >4 μg/ml (43). The decrease in ∆phoPQEcl cell viability suggested that PhoPQEcl signaling is required for colistin heteroresistance. Interestingly, viability was not altered when the ∆pmrABEcl mutant was grown in colistin, which implied that PmrABEcl does not regulate colistin heteroresistance. Furthermore, wild type E. cloacae grown in colistin demonstrated approximately ten-fold less CFUs at hour two (P value <0.05), suggesting that there was a survival defect in early logarithmic growth phase. However, the fitness defect was no longer significant at hour three and by hour four, CFUs were equivalent to growth without colistin (Fig 1A).
Due to reports of colistin heteroresistance in E. cloacae and other ECC strains (35, 40), we subjected wild type, ∆phoPQEcl, ∆phoPQEcl/pPhoPQEcl, and ∆pmrABEcl E. cloacae to colistin E-test strip analysis, which provides a convenient method to observe heteroresistance (Fig S1). Squatter colonies within the zone of inhibition indicated colistin heteroresistance in wild type, ∆phoPQEcl/pPhoPQEcl, and ∆pmrABEcl strains, but not ∆phoPQEcl. We confirmed colistin heteroresistance using population analysis profiling (PAP) (Table 1). Minimal inhibitory concentration (MIC) values were calculated using the broth microdilution (BMD) method (Table 1). Wild type, ∆phoPQEcl/pPhoPQEcl, and ∆pmrABEcl E. cloacae all demonstrated MICs >256 μg/ml, while the ∆phoPQEcl, ∆phoPQEcl/pPhoPQH277A, ∆phoPQEcl/pPhoPD56AQ and ∆arnEcl (arnBCADTEFEcl) MIC was 0.5 μg/ml. Together, these studies confirm that PhoPQEcl signal transduction and the arnEcl biosynthetic operon (L-Ara4N) are required for colistin heteroresistance in E. cloacae.
Since lipid A modifications induce colistin resistance in pathogenic Enterobacteriaceae (5), we analyzed wild type and mutant E. cloacae lipid A for modifications. 32P-radiolabelled lipid A was isolated and chromatographically separated based on hydrophobicity. As controls, we also analyzed lipid A from E. coli strain W3110 (Fig 1B, lane 1), which does not significantly modify its lipid A, and strain WD101(Fig 1B, lane 9), which constitutively expresses pmrA to produce modified lipid A (19). Thin layer chromatography (TLC) analysis indicated that wild type E. cloacae produced a mixture of lipid A consistent with modified and unmodified species (Fig 1B, lane 2). ∆phoPQEcl and the ∆arnEcl strains did not produce modified lipid A (Fig 1B, lanes 3 and 8). PhoPQ complementation fully restored production of modified lipid A in the phoPQ mutant (Fig 1B, lane 4). Furthermore, site-directed mutagenesis to substitute H277 in PhoQEcl or D57 in PhoPEcl with alanine limited lipid A assembly to only unmodified species (Fig 1B lanes 5 and 6). These results confirm that PhoPQEcl phosphotransfer and L-Ara4N biosynthesis are essential for lipid A modification in E. cloacae.
Indirect PhoPQ regulation of lipid A modifications have been described and are conserved among Enterobacteriaceae (5). PhoPQ directly activates PmrD expression, which binds PmrA to induce PmrAB-dependent pEtN and L-Ara4N lipid A modifications (27). In contrast, E. cloacae does not encode a PmrD homolog (44). Furthermore, the pmrABEcl mutant assembled a modified lipid A, similar to wild type (Fig 1B, lane 7), and exhibited colistin heteroresistance (Fig 1A, Table 1, Fig S1A), suggesting that PmrABEcl does not regulate colistin heteroresistance in E. cloacae. Our data contrasts with previous reports describing lipid A modification regulatory pathways in other Enterobacteriaceae, which cannot modify lipid A with L-Ara4N or pEtN when PmrAB signaling is disrupted (19–22, 27).
The lipid A anchor of LPS is a pathogen associated molecular pattern (PAMP) that is bound with high affinity by the mammalian host TLR-4/MD-2 complex (45), which activates a proinflammatory response to clear the bacterial infection (46). Structural alterations to lipid A can dramatically alter TLR-4/MD-2-dependent host immune activation (2) and a previous report nicely demonstrated that E. cloacae colistin heteroresistance was induced by innate immune effectors (40). Therefore, we examined if E. cloacae containing modified or unmodified lipid A would differentially activate TLR-4/MD-2 in a human embryonic kidney reporter cell line (HEK-blue) (2). Wild type and phoPQEcl mutant strains stimulated TLR-4/MD-2-dependent activation equally (Fig S1B), suggesting that lipid A modifications do not significantly alter host immune recognition. Reporter activation by E. cloacae lipid A was attenuated compared to E. coli lipid A at higher cell densities, suggesting differential recognition by the human TLR-4/MD-2 complex. The Gram-positive Staphylococcus aureus did not produce lipid A and did not stimulate the TLR-4/MD-2 complex (Fig S1B). Thus, while PhoPQEcl-dependent lipid A modifications contribute to CAMP resistance in E. cloacae, they do not significantly affect innate immune recognition and reactivity.
Determination of E. cloacae lipid A modifications
In order to define outer membrane modifications in E. cloacae, we isolated lipid A from wild type and ∆phoPQEcl. Purified lipid A was analyzed by direct infusion nanoESI. The MS1 spectra with a range of m/z 750-2000 are shown in Figure S2. The expanded MS1 spectrum (m/z 850-1200) of lipid A isolated from wild type E. cloacae demonstrated three distinct modifications: (i) addition of either one or two L-Ara4N moieties (red), (ii) palmitate (C16:0) addition (green), and (iii) hydroxylation (Fig 2A) The MS1 spectrum of lipid A isolated from ∆phoPQEcl did not produce L-Ara4N modified lipid A (Fig 2B). Hydroxyl addition was not labeled for simplicity, but correlates with a m/z shift of 8 of the doubly-charged molecular ions. Higher-energy collisional dissociation (HCD) and ultraviolet photodissociation (UVPD) MS/MS spectra were obtained for the ions of m/z 1042.68 and 1161.79 from wild type and the ions of m/z 911.62 and 1030.73 from ∆phoPQ E. cloacae (Fig S3, S4, S5 and S6). Analysis of the MS/MS spectra from wild type (m/z 1042.68) indicated PhoPQEcl-dependent addition of L-Ara4N at both the 1- and 4’-phosphates (Fig S3). The MS/MS spectra for the ion of m/z 1161.79 (wild type E. cloacae) showed addition of L-Ara4N at both the 1- and 4’-phosphates and palmitate addition to the R-2-hydroxymyristate (Fig S4). Analysis of lipid A from the phoPQEcl mutant (m/z 911.62) completely lacked L-Ara4N modified lipid A (Fig S5) and analysis of the m/z 1030.73 ion from the phoPQEcl mutant demonstrated that palmitate addition at the R-2-hydroxymyristate position of lipid A occurred independent of PhoPQEcl (Fig S6).
Based on transcriptomics studies, a previous report suggested that E. cloacae adds pEtN and L-Ara4N to lipid A to develop colistin heteroresistance (40). However, our genetic and high resolution mass spectrometry analysis demonstrate that only L-Ara4N modifies the 1- and 4’-phosphates of lipid A in a PhoPQEcl-dependent manner (Fig 2A and B) and this amine-containing modification correlates with colistin heteroresistance (Fig 1A and Table 1).
L-Ara4N lipid A modifications are dependent on PhoPQEcl, but not PmrABEcl
To further characterize lipid A modifications in the ∆pmrABEcl mutant, we analyzed purified lipid A using MALDI-TOF mass spectrometry. While wild type E. cloacae produced a lipid A mixture, which included L-Ara4N modified lipids (Fig S7A and B), analysis of ∆phoPQEcl and ∆arnEcl indicated that L-Ara4N modified lipid A were not present. Expression of PhoPQEcl in trans restored L-Ara4N modified lipid A in the phoPQEcl mutant. Importantly, ∆pmrABEcl produced the L-Ara4N modification, similar to wild type (Fig S7A). The m/z of each prominent peak in our MALDI-MS analysis corresponded with the exact mass of an expected structure with only the L-Ara4N-containing structures demonstrating colistin resistance (Fig S7B). Here, we confirmed that L-Ara4N modification of lipid A in E. cloacae is not dependent on PmrABEcl.
PhoPEcl directly binds to the arnBEcl promoter
The arn operon is composed of seven genes and expression is driven by a promoter upstream of arnB (20). This genetic organization is conserved in E. cloacae as illustrated in Fig 3A. phoP expression is autoregulated in Enterobacteriaceae, where PhoP binds to the PhoP box where it interacts with RNA polymerase to induce transcription (47). The putative PhoP box in the phoP promoter region (PphoP) is conserved in E. coli, Salmonella, and E. cloacae (Fig 3B). Alignment of the E. cloacae arnB promoter region (ParnB) with E. coli, Salmonella, and E. cloacae PphoP suggested a putative PhoP box region. Importantly, E. cloacae ParnB, which encodes a putative PhoP box, is highly conserved among ECC. However, this feature was not encoded within E. coli ParnB, suggesting that regulatory mechanisms that control promoter activation are different (Fig 3B).
We performed electrophoretic mobility shifts (EMSAs) using E. cloacae ParnB to determine if PhoPEcl directly binds the promoter to activate arnEcl transcription. Increasing concentrations of purified PhoPEcl (Fig S8) induced a shift of the biotinylated arnBEcl promoter fragment, which contains the putative PhoP box binding motif (Fig 3C). Importantly, PhoPEcl does not bind to E. coli ParnB, which does not encode the PhoP box motif (Fig 3C). Furthermore, the PhoPEcl-arnBEcl promoter interaction was abrogated when unlabeled E. cloacae ParnB was added in increasing ratios, as a competitive inhibitor. We also show that the interaction is specific because addition of noncompetitive DNA (poly dI-dC) did not reduce the PhoPEcl and E. cloacae ParnB interaction (Fig 3D). Lastly, PhoPEcl bound E. cloacae and E. coli PphoP, which both encode the nucleotide sequence specific to the PhoP box (Fig 3E). Together, these findings suggest that E. cloacae encodes a mechanism that enables L-Ara4N biosynthesis to respond directly to PhoPQEcl.
RNA-sequencing analysis of the phoPQEcl and pmrABEcl mutants
To better understand the PhoPEcl and PmrABEcl regulatory products, we isolated and sequenced total RNA from wild type and mutant E. cloacae strains. A heat map illustrates the fold change of arnEcl, phoPQEcl, and pmrABEcl gene expression in the TCS mutants relative to wild type (Fig 4). Expression of the arnEcl genes were significantly down regulated in ∆phoPQEcl compared to wild type, suggesting that activation of the pathway is dependent on PhoPQEcl. In contrast, arnEcl gene expression was not significantly altered in the ∆pmrABEcl mutant relative to wild type.
Colistin resistance increases within the E. cloacae population in response to limiting Mg2+
Together the transcriptomic analysis (Fig 4) and colistin heteroresistance in wild type E. cloacae (Fig 1A and Table 1) suggest constitutive addition of L-Ara4N to lipid A in a subset of the population under standard growth conditions. In E. coli and Salmonella, PhoPQ is activated by various signals, including low Mg2+ and CAMPs (13, 23, 25). PhoP activates PmrAB, which stimulates pEtN and L-Ara4N addition to lipid A (27). Here we analyzed if PhoPQEcl responds to similar physiological cues to induce colistin resistance in E. cloacae. Wild type and mutant E. cloacae were grown in N minimal medium with high (10 mM) or low (10 μM) Mg2+ levels. All cultures were exposed to colistin at mid-logarithmic growth. Wild type and complemented phoPQEcl mutant strains grown in high Mg2+ demonstrated some susceptibility to 5 and 10 μg/ml of colistin (Fig 5A, High Mg2+), suggesting colistin-susceptible and-resistant populations were present. When grown under limiting Mg2+ conditions, E. cloacae cells were resistant (Fig 5A, Low Mg2+). The phoPQEcl mutant demonstrated a fitness defect in either Mg2+ concentration when exposed to colistin (Fig 5A). These data suggest that PhoPQEcl induces colistin resistance in response to limiting Mg2+ growth conditions.
PhoPQEcl responds to limiting Mg2+ conditions by inducing L-Ara4N lipid A modification
To determine if increased colistin resistance was dependent on L-Ara4N modification of lipid A, we isolated lipid A after growth in either low or high Mg2+. TLC analysis demonstrated that wild type and the complemented phoPQEcl mutant primarily produced L-Ara4N-modified lipid A when Mg2+ concentrations were limiting (Fig 5B, Low Mg2+). In contrast, the same strains grown in excess Mg2+, produced a mixture of modified and unmodified lipid A (Fig 5B, High Mg2+). Interestingly, growth in excess Mg2+ does not completely shut-off production of PhoPQEcl-dependent lipid A modification in E. cloacae, as was previously shown in E. coli (27). We did observe a decrease in the relative amount of the di-L-Ara4N form while increasing the ratio of unmodified lipid A species (Fig 5B). Together, these studies suggest that a subset of the clonal E. cloacae population activates PhoPQEcl-dependent L-Ara4N of lipid A under standard growth conditions. However, depletion of Mg2+ induces PhoPQEcl signaling to enhance L-Ara4N modification (Fig 5B) and colistin resistance (Fig 5A) throughout the population.
Discussion
E. cloacae and other ECC members encode PmrABEcl and PhoPQEcl homologs, which we hypothesized would function in a signaling pathway to regulate L-Ara4N and pEtN modification of lipid A based on previous transcriptomics analysis of resistant and susceptible populations (40) and because these lipid A modifications are highly conserved among Enterobacteriaceae (5). However, our genetic and high-resolution mass spectrometry analysis of E. cloacae lipid A determined that colistin heteroresistance in E. cloacae was mediated by PhoPQEcl-dependent L-Ara4N lipid A modification. Therefore, we have identified a mechanism of ECC colistin heteroresistance that involves the PhoPQ system.
E. cloacae and other ECC members do not encode a PmrD homolog, which couples PhoPQ signal transduction to regulation of PmrA-dependent genes in many Enterobacteriaceae (5). Moreover, PmrAEcl shares only 52% identity with E. coli PmrA and PmrBEcl shares only 57% identity with E. coli PmrB, suggesting the L-Ara4N lipid A modification pathway in E. cloacae diverged from other Enterobacteriaceae. We confirmed direct binding of PhoPEcl to the arnBEcl promoter, which supports a model where L-Ara4N addition to lipid A and colistin heteroresistance in E. cloacae is dependent on PhoPQEcl, but not PmrABEcl.
Research from other groups has outlined a complex regulatory network in E. coli and Salmonella that tightly regulates lipid A L-Ara4N and EptA modifications (19–22, 27). We hypothesize that uncoupling PmrABEcl signal transduction from L-Ara4N modification bypasses a key regulatory checkpoint, which likely promotes constitutive arnEcl transcription and L-Ara4N modification of lipid A in a subset of the clonal E. cloacae population. Since selection has driven E. cloacae and other ECC to maintain an altered lipid A modification signaling network, we predict that it is advantageous to maintain a CAMP resistant subpopulation in some environments. Presumably, the alternative regulatory mechanism promotes bacterial fitness in environments specific to its commensal and pathogenic niches.
While the dynamics of the regulatory cascades that control lipid A modification in Gram-negative bacteria have been extensively characterized in E. coli and Salmonella and generalized across Enterobacteriaceae, mechanisms that regulate lipid A modifications in E. cloacae highlight variations that promote clinically important resistance levels. Furthermore, colistin heteroresistance has also been associated with Klebsiella pneumoniae (48), another Enterobacteriaceae family member, which highlights the importance of studying antimicrobial resistance mechanisms at the species and strain level.
Materials and Methods
Bacterial Strains and Growth
E. cloacae subsp. cloacae ATCC 13047 and ECC strains were initially grown from freezer stocks on Luria-Bertani (LB) agar. Isolated colonies were used to inoculate LB broth or N minimal medium (0.1M Bis-Tris, pH 7.5 or 5.8, 5 mM KCl, 7.5 mM (NH4)2SO4, 0.5 M K2SO4, 1 mM KH2PO4, 0.10% casamino acids 0.2% glucose, 0.0002% thiamine, 15 μM FeSO4, 10 μM or 10 mM MgSO4) at 37° C. Kanamycin was used at 25 μg/ml for selection and colistin was used at 5 μg/ml or 10 μg/ml where indicated.
All strains and plasmids used in this study are listed in Table S1. Briefly, E. cloacae subsp. cloacae 13047 mutant strains were constructed as previously described using recombineering with the plasmid pKOBEG (49). Linear PCR products were introduced in to the E. cloacae ATCC 13047/pKOBEG strain by electroporation and plated on selective media. Selected clones were transformed with pCP20 to cure the antibiotic resistance cassette.
To complement E. cloacae mutants, the coding sequence from phoPQEcl was cloned into the SalI and KpnI sites in pMMBKn (3). To generate point mutants in PhoQH277A and PhoQD56A, site directed mutagenesis was performed using Pfu Turbo using primers that incorporated the associated alanine-encoded nucleotide replacement. All constructs were validated using Sanger sequencing. IPTG inducible constructs were transformed into the phoPQ mutant and grown in 2.0 mM IPTG to induce expression.
Colony Forming Unit Counts
E. cloacae subsp. cloacae 13047 and mutant strains were initially grown from freezer stocks on Luria-Bertani (LB) agar. Isolated colonies were resuspended and used to inoculate LB broth with 10 μg/ml or without colistin at an OD600 = 0.01. Cells were plated at designated time points on LB agar. Plates were grown overnight at 37° C and colony forming units (CFU) were counted and reported.
Broth Microdilution assays
MICs of colistin were determined in triplicate by the broth microdilution (BMD) method. Briefly strains were inoculated from overnight cultures at an OD600 = 0.1. Various concentrations (0 - 256 μg/ml) of colistin were added to each well and cultures were incubated overnight. Growth was indicated by a reading the OD600 and the lowest concentration at which growth was inhibited was recorded as the MIC. E. coli W3110 and WD101 were used as control strains. In some cases, ‘skip wells’ were observed suggesting a heteroresistance phenomenon and the MIC was determined disregarding the clear wells (35).
Population Analysis
Population analysis profiling was performed by plating a high inoculum (1 X 1010 CFU) onto LB agar containing 1 to 64 μg/ml colistin (in 2-fold increments). Plates were incubated overnight at 37° C and frequency of the subpopulation was determined by dividing by the total number of cells (50).
Isolation of Lipid A
Isolation of lipid A for TLC analysis involved 32P-radiolabeling of whole cells was performed as previously described with slight modifications (51). In brief, 12.5 ml of E. cloacae was grown at 37° C to OD600 = 1.0. Bacteria were harvested by centrifugation at 10,000 X g for 10 min. Lipid A extraction was carried out by mild-acid hydrolysis as previously described (52).
Mass Spectrometry
MS1 spectra of lipid A in Figure 4 were collected on a MALDI-TOF/TOF (Axima Performance, Shimadzu) mass spectrometer in the negative mode. All other spectra were collected in the negative mode on a Thermo Scientific Orbitrap Fusion Lumos mass spectrometer (San Jose, CA, USA) modified with a Coherent ExciStar XS ArF excimer laser (Santa Clara, CA), as previously described (53). HCD was performed with the normalized collision energy (NCE) of 25%. UVPD was performed with the laser emitting 193 nm photons at 5 mJ per laser pulse with 5 pulses per scan. The laser pulse repetition rate was 500 Hz. The instrument was operated at 120000 resolving power with a precursor isolation window of 3 m/z. All samples were dissolved in 50:50 MeOH:CHCl3 and directly infused into the mass spectrometer via a static nano-electrospray ionization source. The presented spectra are an average of 50 scans.
TLR-4 Signaling Assays
HEK-Blue hTLR4, cell line was maintained according to the manufacturer specifications (Invivogen). Overnight bacterial cultures in stationary phase were serial diluted for assays as previously described (2, 3). At least two biological replicates were each done in triplicate and one representative set was shown.
Colistin Survival Assays
Polymyxin E survival assay analysis were performed as previously described with slight modifications (27). Wild type and mutant E. cloacae strains were grown overnight on LB agar. The following day, media N minimal medium with pH = 7.5, 10 μM MgSO4 were inoculated at OD600 = 0.1 with bacteria from overnight cultures that were washed with N minimal media without Mg2+ or iron. Cultures were grown until OD600 = 0.6, when they were split and treated with 0, 5 or 10 μg/ml of colistin (Polymyxin E). Cultures were incubated for 1 h at 37° C and then colony-forming units were plated and calculated. Percent survival was calculated by dividing the number of bacteria after treatment with colistin relative to those incubated in the absence of colistin and then multiplied by 100.
Protein Purification
To purify the PhoPEcl protein, the coding sequence was cloned into pT7-7Kn, as previously described (7). Briefly the phoPEcl CDS was amplified from E. cloacae cDNA with primers that added a C-terminal His8X tag. From an overnight starter culture, 1 Liter of LB broth containing 25 μg/ml of kanamycin was inoculated at 1:50 and grown at 37° C until the OD600 = 0.5. IPTG was added to a final concentration of 1mM, and the culture was incubated at 37° C for an additional 4 h. Bacteria were recovered by centrifugation at 10,000 × g for 10 min, and the bacteria were resuspended in lysis buffer. Bacteria were lysed using sonication and the soluble fraction was recovered by centrifugation at 10,000 × g for 30 min. PhoPEcl-His8X was purified on a Ni-nitrilotriacetic acid (NTA) beads according to the manufactures instructions (Qiagen).
Electrophoretic Mobility Shift Assay
PhoPEcl-His8X proteins were purified as described above. EMSAs were performed based on a modified protocol (6). 250-bp DNA fragments of phoPEcl and arnBEcl spanning −230 to +20 relative to the translational start site were amplified from E. cloacae or E. coli cDNA using 5’-biotinylated primers. PhoPEcl-His8X proteins were incubated with biotinylated DNA at 25° C for 20 min. For competition experiments, unlabeled E. cloacae ParnB and poly (dI-dC) were added at 1:1, 2:1, or 5:1 ratios relative to biotin-labeled ParnB DNA, and 0.1 - 10 μM of PhoPEcl-His8X proteins were used. After electrophoresis at 4° C, protein/DNA was transferred onto a positively charged nylon membrane. Blots were blocked in 5% milk in TBS for 20 min and streptavidin conjugated HRP was used at a 1:300 dilution.
Nucleic Acid Extraction
Total RNA was extracted using the Direct-Zol RNA MiniPrep Kit (Zymo Research) from E. cloacae grown to a final OD600 = 0.6. Isolated RNA was treated with DNA-free DNA removal kit (Thermo-Fisher Scientific) to eliminate genomic DNA contamination. DNase-depleted RNA was used for qRT-PCR and RNA-seq.
RNA-sequencing
RNA-sequencing was performed as previously described (54). Briefly, DNA-depleted RNA was processed for Illumina sequencing using the NEB Next Ultra Directional RNA Library Prep kit for Illumina as described by the manufacturer (NEB). Sequencing was performed using Illumina HiSeq. Sequencing data was aligned to the E. cloacae subs. cloacae ATCC 13047 published genome annotations (44) using CLC genomic workbench software (Qiagen) and RPKM expression values were determined. The weighted proportions fold change of expression values between samples was determined and a Baggerley’s test on proportions was used to generate a false discovery rate corrected P-value. We then used a cut-off of 2-fold weighted proportions absolute change with a false-discovery rate corrected P-value of ≤ 0.05 to identify significantly differentially regulated genes between samples. The sequencing data for the clinical isolates has been deposited in the Nation Center for Biotechnology’s Gene Expression Omnibus (PRJNA461875).
Acknowledgements
This work was supported by NIH GM103655 (J.S.B), and Welch Foundation F-1155 (J.S.B.). Funding from the UT System for support of the UT System Proteomics Core Facility Network is gratefully acknowledged.
We would like to thank Cara Boutte and Mark Pellegrino for thoughtful review of the manuscript.