lpxD Genes Research Articles

Genetic alterations in the pmrAB two-component system and lipid A biosynthesis genes of polymyxin-resistant Acinetobacter baumannii isolates.

The rate of pandrug-resistant Acinetobacter baumannii strains is on the rise in all continents. This bacterium can acquire resistance to all antibiotics, even to colistin. Alterations in the lipid A or/and the two-component pmrAB were earlier detected in colistin resistance. We investigated and analyzed two strains of A. baumannii (ABRC1 and ABRC2) isolated from two patients admitted to intensive care unit with a septic shock. Both strains were resistant to all tested antibiotics including colistin with a MIC >256 mg L-1. Colistin resistance genes (pmrA, pmrB, lpxA, lpxC, lpxD, and lpsB) of two strains (ABRC1 and ABRC2) were investigated by PCR and sequencing. Obtained nucleic acid sequences were aligned with reference sequences of ATCC 19606 and 17987. In this study two amino acid mutations, N287D in the lpxC gene and E117K in the lpxD gene, were detected in both ABRC1 and ABRC2 strains. ABRC1 had an additional H200L mutation in the pmrA gene. Both colistin resistant strains harbored the same A138T mutation in the pmrB gene. The ABRC2 strain also had an alteration in the kinase domain, specifically an R263S substitution of the histidine kinase domain. Three identical mutations were found in the lpsB gene of both A. baumannii strains: Q216K + H218G + S219E. As a result, a newly deduced protein sequence in both ABRC1 and ABRC2 strains differed from those described in ATCC 17978 and 19606 strains was determined. Colistin resistance is multifactorial in A. baumannii. In our study we detected novel mutations in colistin resistant A. baumannii clinical isolates.

Prevalence and molecular mechanisms of colistin resistance in Acinetobacter baumannii clinical isolates in Tehran, Iran.

Colistin is one of the last remaining active antibiotics against multidrug resistant Gram-negative bacteria. However, several recent studies reported colistin-resistant (ColR) Acinetobacter baumannii from different countries. In the current study, we investigated molecular mechanisms involved in colistin resistance in A. baumannii isolates from different clinical samples.A total of 110 clinical A. baumannii isolates were collected from two hospitals in Tehran. Minimum inhibitory concentrations (MICs) were determined by broth microdilution according to the Clinical and Laboratory Standards Institute. For the ColR isolates, mutation was detected in pmrA, pmrB, lpxA, lpxC, and lpxD genes using the polymerase chain reaction (PCR) and sequencing. Moreover, the relative expression of the pmrC gene was calculated using quantitative reverse transcription PCR. Three colistin resistant isolates were identified with MIC between 8 and 16 μg/mL and were resistant to all the tested antimicrobial agents. All the three isolates had a mutation in the pmrB, pmrA, lpxA, lpxD, and lpxC genes. Moreover, the overexpression of pmrC gene was observed in all isolates. Our results showed that the upregulation of the PmrAB two component system was the primary mechanism linked to colistin resistance among the studied colistin resistant A. baumannii isolates.

Acquisition of Colistin Resistance Links Cell Membrane Thickness Alteration with a Point Mutation in the lpxD Gene in Acinetobacter baumannii.

Acinetobacter baumannii is one of the most common causes of nosocomial infections in intensive care units. Its ability to acquire diverse mechanisms of resistance limits the therapeutic choices for its treatment. This especially concerns colistin, which has been reused recently as a last-resort drug against A. baumannii. Here, we explored the impact of gaining colistin resistance on the susceptibility of A. baumannii to other antibiotics and linked colistin resistance acquisition to a gene mutation in A. baumannii. The susceptibility of 95 A. baumannii isolates revealed that 89 isolates were multi-drug resistance (MDR), and nine isolates were resistant to colistin. Subsequently, three isolates, i.e., MS48, MS50, and MS64, exhibited different resistance patterns when colistin resistance was induced and gained resistance to almost all tested antibiotics. Upon TEM examination, morphological alterations were reported for all induced isolates and a colistin-resistant clinical isolate (MS34Col-R) compared to the parental sensitive strains. Finally, genetic alterations in PmrB and LpxACD were assessed, and a point mutation in LpxD was identified in the MS64Col-R and MS34Col-R mutants, corresponding to Lys117Glu substitution in the lipid-binding domain. Our findings shed light on the implications of using colistin in the treatment of A. baumannii, especially at sub-minimum inhibitory concentrations concentrations, since cross-resistance to other classes of antibiotics may emerge, beside the rapid acquisition of resistance against colistin itself due to distinct genetic events.

Antimicrobial and Anti-Biofilm Effect of an Electrolyzed Superoxidized Solution at Neutral-pH against Helicobacter pylori.

The presence of Helicobacter pylori in the oral cavity has been associated to the failure of antimicrobial therapy in patients with gastrointestinal infection and the development of oral diseases. However, it has been reported that the maintenance of good oral hygiene can improve the therapeutic success rates, where the use of mouthwashes with anti-Helicobacter activity would help to achieve it. The aim was to evaluate the antimicrobial activity of OxOral® mouthwash against H. pylori and its effect on biofilm formation. The minimum inhibitory concentration (MIC) of OxOral® (pH = 6.4–7.5, ORP = 650–900 mV) against H. pylori was calculated testing serial dilutions 0.117–15 ppm against 1 × 108 CFU/mL of H. pylori (ATCC® 700824™) by broth microdilution method using 96‐well plates. The H. pylori biofilm formation was determined by the optical density measurement at 600 nm from coverslips stained with 0.1% crystal violet. The gene expression of ureA, luxS, flaA, omp18, and lpxD were analyzed by RT‐qPCR. OxOral® cytotoxicity was evaluated in a human gingival fibroblast cell line by MTT assay. MIC was of 3.75 ppm, with 99.7 ± 7.7% bacterial growth inhibition. In the negative control, the biofilm formation was observed, whereas when bacteria were treated with OxOral® at 0.234, 0.469, and 0.938 ppm, an inhibition of 35.5 ± 0.9%, 89.1 ± 1.2%, and 99.9 ± 5.5% were obtained, respectively. The gene expression analysis showed that flaA, omp18, and lpxD genes were down‐regulated with OxOral® compared with control (p < 0.05). Low cytotoxicity of 16.5 ± 7.6% was observed at the highest dose (15 ppm); no significant differences were observed from 15 to 0.469 ppm compared to the control of untreated cells (p > 0.05). Our results reveal an important anti-Helicobacter activity of OxOral® and open the possibility of its therapeutic use new studies, which would increase the success rate of conventional therapies against H. pylori.

LpxD gene knockout elicits protection to Litopenaeus vannamei, white shrimp, against Vibrio parahaemolyticus infection

Vibrio parahaemolyticus is a Gram-negative bacterium causing acute hepatopancreatic necrosis disease (AHPND) in white shrimp, Litopenaeus vannamei. LpxD gene, a principal component of lipid A, which plays a vital role in the biosynthesis of lipopolysaccharides (LPSs) responsible for the pathogenesis of Gram-negative bacteria However, the function of lpxD in V. parahaemolyticus’ virulence has not been studied. In this study, lpxD gene knockout and its role in V. parahaemolyticus’ virulence in white shrimp were determined. The lpxD gene was knocked out by homologous recombination. Virulence and immunogenicity of the lpxD gene was disrupted and the pirA, the major virulence factor of AHPND, and AP1 gene were intact in white shrimp. White shrimp fed with normal diet with VP△lpxD displayed protection (reduced mortality rate from 66 to 27%) after challenge with V. parahaemolyticus. Our results indicated that knockout of the lpxD gene can cause attenuation of V. parahaemolyticus in white shrimp, and VP△lpxD could be used as a live attenuated oral vaccine against V. parahaemolyticus in white shrimp.

Promoters of Colistin Resistance in Acinetobacter baumannii Infections.

Objectives: We aimed to describe the mechanisms of colistin resistance in Acinetobacter baumannii. Materials and Methods: Twenty-nine patients diagnosed with colistin-resistant A. baumannii infection were included to the study. The mutations in pmrCAB, lpxA, lpxC, and lpxD genes, expression of pmrCAB, carbapenemases, and mcr-1 positivity were studied. Results: Twenty-seven (93%) of the patients received IV colistin therapy during their stay, and the case fatality rate was 45%. All mutations in pmrC and pmrB were found to be accompanied with a mutation in lpxD. The most common mutations were I42V and L150F in pmrC (65%), E117K in lpxD (65%), and A138T in pmrB (58.6%). The colistin minimum inhibitory concentrations (MICs) of the isolates having any of these four mutations were higher than the isolates with no mutations (p < 0.001). The two most common mutations in pmrC (I42V and L150F) were found to be associated with higher expressions of pmrA and pmrC and higher colistin MIC values (p = 0.010 and 0.031). All isolates were blaOXA-23 positive. Conclusion: Coexistence of the lpxD mutation along with mutations in pmrCAB indicates synergistic function of these genes in development of colistin resistance in A. baumannii.

Immunization with lipopolysaccharide-free outer membrane complexes protects against Acinetobacter baumannii infection

Outer membrane complex (OMC) vaccines, which contain antigens from the bacterial outer membrane, have been developed for multiple Gram-negative bacteria. However, OMC vaccines demonstrate high endotoxin activity due to the presence of lipopolysaccharide in the bacterial outer membrane, thus precluding their use in humans. We isolated OMCs from an LPS-deficient strain of A. baumannii (IB010) which completely lacks LPS due to a mutation in the lpxD gene. OMCs from IB010 demonstrated a more than 10,000-fold reduction in endotoxin activity compared to OMCs from wild type A. baumannii. Vaccination with IB010 OMCs produced similar levels of antigen-specific IgG and IgM after two administrations compared to wild type OMCs, and resulted in a similar reduction in post-infection spleen bacterial loads and serum pro-inflammatory cytokine levels. Vaccination with IB010 OMCs provided significant protection against infection compared to control mice, indicating the LPS-free OMCs could contribute to vaccine strategies for preventing infection by A. baumannii.

Phenotypic changes associated with Colistin resistance due to Lipopolysaccharide loss in Acinetobacter baumannii

ABSTRACTAcinetobacter baumannii can acquire resistance to colistin via complete loss of lipopolysaccharide (LPS) biosynthesis due to mutations in the lpxA, lpxC and lpxD genes. However, although colistin is increasingly being used for the treatment of multidrug resistant infections, very few A. baumannii clinical isolates develop colistin resistance through loss of LPS biosynthesis. This may suggest that LPS loss affects virulence traits that play a role in the transmission and pathogenesis of A. baumannii. In this study we characterize multiple virulence phenotypes of colistin resistant, LPS-deficient derivatives of the ATCC 19606 strain and five multidrug resistant clinical isolates and their colistin resistant, LPS-deficient derivatives. Our results indicate that LPS loss results in growth defects compared to the parental strain in vitro both in laboratory media and human serum (competition indices of 0.58 and 7.0 × 10−7, respectively) and reduced ability to grow and disseminate in vivo (competition index 6.7 × 10−8). Infection with the LPS-deficient strain resulted in lower serum levels of pro-inflammatory cytokines TNF-α and IL-6 compared to the parent strain, and was less virulent in a mouse model of disseminated sepsis. LPS loss also significantly affected biofilm production, surface motility, growth under iron limitation and susceptibility to multiple disinfectants used in the clinical setting. These results demonstrate that LPS loss has a significant effect on multiple virulence traits, and may provide insight into the low incidence of colistin resistant strains lacking LPS that have been reported in the clinical setting.

Molecular Mechanisms of Colistin Resistance Among Pandrug-Resistant Isolates of Acinetobacter baumannii with High Case-Fatality Rate in Intensive Care Unit Patients.

Colistin is considered a last-hope antibiotic against extensively drug-resistant isolates of Acinetobacter baumannii. Resistance to colistin has been rarely reported for A. baumannii. Genetic alterations in the PmrA-PmrB two-component system and lipid A biosynthesis genes may be associated with colistin resistance. We investigated molecular mechanisms of colistin resistance in three clinical colistin-resistant (ColR) and two colistin-susceptible (ColS) A. baumannii isolates. A ColR mutant was generated in vitro by repetitive drug exposure. The pmrA, pmrB, lpxA, lpxC, and lpxD genes were amplified and sequenced. To evaluate association between colistin resistance and upregulation of pmrCAB operon, transcriptional level of the pmrC gene encoding for lipid A phosphoethanolamine (PEtN) transferase was quantified by reverse transcription quantitative PCR (RT-qPCR) analysis. All clinical and in vitro-selected ColR isolates harbored at least one point mutation in the pmrB gene, including A142V, P233S, T235I, and A227V substitutions as well as duplication of H325. No alteration was found in the pmrA and other amino acid substitutions identified in the pmrB as well as lpx genes did not seem to be involved in colistin resistance as they were found in both ColS and ColR isolates. RT-qPCR analysis revealed a correlation between colistin resistance and pmrC overexpression. Specific alterations in the PmrB, linked to overproduction of PEtN transferase, triggered colistin resistance in the studied A. baumannii isolates.

The role of LpxA/C/D and pmrA/B gene systems in colistin-resistant clinical strains of Acinetobacter baumannii

ObjectiveTo study LpxA/C/D and pmrA/B gene systems role in colistin resistant clinical strains of Acinetobacter baumannii. MethodsTransmission Electron Microscopy (TEM) was employed to observe changes in the cell wall, inner and outer membranes. GC-Chromatography was applied to quantify the fatty acid content as a result of changes in the LPS in clinical A. baumannii strains. Furthermore, the isolates were subjected to molecular biology approaches employing Real-Time PCR to evaluate the mRNA expression levels of pmrA and pmrB. Colistin-resistant and colistin-dependent A. baumannii isolates were further screened by PCR amplification to determine mutations in lpxA, lpxC and lpxD genes responsible for lipid A biosynthesis. ResultsTransmission Electron microscopy of six A. baumannii isolates showed that 2 colistin-resistant (Col-R) and 2 colistin-dependent (Col-D) A. baumannii had decreased integrity of the outer and inner membrane and lost uniformity in the periplasmic space compared with 2 susceptible (Col-S) Acinetobacter baumannii strains. GC-Chromatography indicated that there was a trend of decreased saturated and unsaturated fatty acid biosynthesis, especially long carbon chain (16:0, 17:0 and 18:0 carbon chains) and almost no alcohol substitution on the low carbon chain fatty acid (increased modification on the long chain fatty acid and the loss of most unidentified fatty acid peaks) in Col-D and Col-R strains in comparison with Col-S and ATCC19606 strains. The expression data from RT-PCR of PmrA/B two-component regulatory system suggest that upregulated gene expression in 4 Col-R and 3 Col-D strains may lead to a modification in and/or loss of lipid A. Lipid A biosynthesis genes sequencing results revealed deletion of 11bp nucleotides and change of one nucleotide in lpxA, and a nucleotide point mutation and insertion in lpxC and lpxD of Col-D and Col-R strains resulting in defective lipid A production and outer membrane lipid synthesis. ConclusionMechanisms of colistin resistance in clinical strains of A. baumannii show that colistin may not serve as an antibiotic of the last resort for treating MDR A. baumannii infections when other antibiotics are ineffective. The mechanisms of colistin resistance should provide an impetus for future research on the development of newer alternative therapies to treat emerging MDR A. baumannii.

Protection of rainbow trout against yersiniosis by lpxD mutant Yersinia ruckeri.

Yersinia ruckeri is a Gram negative bacteria causing yersiniosis in freshwater and marine fish. Lipid A, important for pathogenesis of Gram negative bacteria, biosynthesis pathway requires nine enzyme catalyzed steps. Although there are nine genes encoding lipid A biosynthesis in bacteria, biosynthesis of lipopolysaccharides relies on lpxD gene that encodes the third pathway enzyme. The roles of LpxD in Y.ruckeri virulence have not been studied. In the present study, in-frameshift deletion of lpxD gene and their role in Y.ruckeri virulence in rainbow trout were determined. For this purpose, 92% of the Y.ruckeri lpxD genes were deleted by homologous recombination. After running in SDS-PAGE and staining with silver stain, no LPS was detectable in the Y.ruckeri ΔlpxD mutant. Virulence and immunogenicity of the Y.ruckeri ΔlpxD mutant (YrΔlpxD) were determined in rainbow trout. Rainbow trout immunized with YrΔlpxD with immersion, or intraperitoneal injection method displayed superior protection (relative percentage survival≥84%) after exposure to wild type Y.ruckeri. In conclusion, our results indicated that deletion of the lpxD gene causes significant attenuation of Y.ruckeri in rainbow trout, and LPS deficient YrΔlpxD could be used as a live attenuated vaccine against Y.ruckeri in rainbow trout. This vaccine can protect fish and it can be applied to fish with different methods such as immersion or injection.

Comparative Genomics of Two ST 195 Carbapenem-Resistant Acinetobacter baumannii with Different Susceptibility to Polymyxin Revealed Underlying Resistance Mechanism.

Acinetobacter baumannii is a Gram-negative nosocomial pathogen of importance due to its uncanny ability to acquire resistance to most antimicrobials. These include carbapenems, which are the drugs of choice for treating A. baumannii infections, and polymyxins, the drugs of last resort. Whole genome sequencing was performed on two clinical carbapenem-resistant A. baumannii AC29 and AC30 strains which had an indistinguishable ApaI pulsotype but different susceptibilities to polymyxin. Both genomes consisted of an approximately 3.8 Mbp circular chromosome each and several plasmids. AC29 (susceptible to polymyxin) and AC30 (resistant to polymyxin) belonged to the ST195 lineage and are phylogenetically clustered under the International Clone II (IC-II) group. An AbaR4-type resistance island (RI) interrupted the comM gene in the chromosomes of both strains and contained the blaOXA−23 carbapenemase gene and determinants for tetracycline and streptomycin resistance. AC29 harbored another copy of blaOXA−23 in a large (~74 kb) conjugative plasmid, pAC29b, but this gene was absent in a similar plasmid (pAC30c) found in AC30. A 7 kb Tn1548::armA RI which encodes determinants for aminoglycoside and macrolide resistance, is chromosomally-located in AC29 but found in a 16 kb plasmid in AC30, pAC30b. Analysis of known determinants for polymyxin resistance in AC30 showed mutations in the pmrA gene encoding the response regulator of the two-component pmrAB signal transduction system as well as in the lpxD, lpxC, and lpsB genes that encode enzymes involved in the biosynthesis of lipopolysaccharide (LPS). Experimental evidence indicated that impairment of LPS along with overexpression of pmrAB may have contributed to the development of polymyxin resistance in AC30. Cloning of a novel variant of the blaAmpC gene from AC29 and AC30, and its subsequent expression in E. coli also indicated its likely function as an extended-spectrum cephalosporinase.

An Amphipathic Undecapeptide with All d-Amino Acids Shows Promising Activity against Colistin-Resistant Strains of Acinetobacter baumannii and a Dual Mode of Action.

Multiple strains of Acinetobacter baumannii have developed multidrug resistance (MDR), leaving colistin as the only effective treatment. The cecropin-α-melittin hybrid BP100 (KKLFKKILKYL-NH2) and its analogs have previously shown activity against a wide array of plant and human pathogens. In this study, we investigated the in vitro antibacterial activities of 18 BP100 analogs (four known and 14 new) against the MDR A. baumannii strain ATCC BAA-1605, as well as against a number of other clinically relevant human pathogens. Selected peptides were further evaluated against strains of A. baumannii that acquired resistance to colistin due to mutations of the lpxC, lpxD, pmrA, and pmrB genes. The novel analogue BP214 showed antimicrobial activity at 1 to 2 μM and a hemolytic 50% effective concentration (EC50) of >150 μM. The lower activity of its enantiomer suggests a dual, specific and nonspecific mode of action. Interestingly, colistin behaved antagonistically to BP214 when pmrAB and lpxC mutants were challenged.

Loss of LPS is involved in the virulence and resistance to colistin of colistin-resistant Acinetobacter nosocomialis mutants selected in vitro.

Acinetobacter nosocomialis has increasingly been reported as an opportunistic pathogen causing nosocomial infections. Although it is more susceptible to all antimicrobial agents than Acinetobacter baumannii, MDR clinical isolates have also been described. In addition, several studies have shown a high percentage of resistance to colistin. Therefore, in the present study we investigated the mechanism of resistance to colistin in this microorganism. Colistin-resistant strains were selected from the original colistin-susceptible A. nosocomialis strain following multi-step mutant selection. Comparative genomic and proteomic analyses of both colistin-susceptible and colistin-resistant A. nosocomialis strains were performed. In addition, virulence was investigated using the Caenorhabditis elegans assay. The colistin-resistant mutants selected showed a lower resistance profile for other types of antibacterial agents together with a significant decrease in virulence. The LT50 (i.e. time required to kill 50% of the nematodes) for the colistin-susceptible strain (WT) was 7 days compared with 9 days for the colistin-resistant strain (256) (P < 0.0001). In the genomic studies, several mutations were observed in the lpxD genes, leading to the loss of LPS in the colistin-resistant strains. The proteomic studies showed several up- and down-regulated proteins that may be involved in colistin resistance or in a decrease in the resistance profile for several antibiotics. This study shows that the mechanism of resistance to colistin by A. nosocomialis is mainly associated with the loss of LPS due to mutations in the lpxD gene, although changes in the expression of some proteins cannot be ruled out. In addition, the acquisition of colistin resistance is related to a decrease in virulence.

ISAba15Inserted into Outer Membrane Protein GenecarOinAcinetobacter baumannii

We identified ISAba15 inserted into the carO gene in an Acinetobacter baumannii isolate. The insert disrupted the lpxD gene, resulting in colistin resistance in A. baumannii. Persistence in carbapenem resistance in A. baumannii isolates with an intact carO gene indicates that loss of the encoded CarO may play a minor role in carbapenem resistance.

Immunization with lipopolysaccharide-deficient whole cells provides protective immunity in an experimental mouse model of Acinetobacter baumannii infection.

The increasing clinical importance of infections caused by multidrug resistant Acinetobacter baumannii warrants the development of novel approaches for prevention and treatment. In this context, vaccination of certain patient populations may contribute to reducing the morbidity and mortality caused by this pathogen. Vaccines against Gram-negative bacteria based on inactivated bacterial cells are highly immunogenic and have been shown to produce protective immunity against a number of bacterial species. However, the high endotoxin levels present in these vaccines due to the presence of lipopolysaccharide complicates their use in human vaccination. In the present study, we used a laboratory-derived strain of A. baumannii that completely lacks lipopolysaccharide due to a mutation in the lpxD gene (IB010), one of the genes involved in the first steps of lipopolysaccharide biosynthesis, for vaccination. We demonstrate that IB010 has greatly reduced endotoxin content (<1.0 endotoxin unit/106 cells) compared to wild type cells. Immunization with formalin inactivated IB010 produced a robust antibody response consisting of both IgG1 and IgG2c subtypes. Mice immunized with IB010 had significantly lower post-infection tissue bacterial loads and significantly lower serum levels of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6 compared to control mice in a mouse model of disseminated A. baumannii infection. Importantly, immunized mice were protected from infection with the ATCC 19606 strain and an A. baumannii clinical isolate. These data suggest that immunization with inactivated A. baumannii whole cells deficient in lipopolysaccharide could serve as the basis for a vaccine for the prevention of infection caused by A. baumannii.

Expression of the lipopolysaccharide biosynthesis gene lpxD affects biofilm formation of Pseudomonas aeruginosa.

Bacterial biofilms are an important cause of nosocomial infections. Microorganisms such as Pseudomonas aeruginosa colonize biotic and abiotic surfaces leading to chronic infections that are difficult to eradicate. To characterize novel genes involved in biofilm formation, we identified the lpxD gene from a transposon-mutant library of P. aeruginosa. This gene encodes a glucosamine-N acyltransferase, which is important for lipopolysaccharide biosynthesis. Our results showed that a loss-of-expression mutant of lpxD was defective for biofilm formation on biotic and abiotic surfaces. Additionally, this mutant strain exhibited significantly decreased bacterial attachment to cultured airway epithelial cells, as well as increased bacterial cytotoxicity toward airway cells. However, consistent with a defect in lipid A structure, airway cells incubated with the lpxD mutant or with mutant lipid A extracts exhibited decreased IL-8 production and necrosis, respectively. Overall, our data indicate that manipulating lpxD expression may influence P. aeruginosa's ability to establish biofilm infections.

LPS remodeling is an evolved survival strategy for bacteria.

Maintenance of membrane function is essential and regulated at the genomic, transcriptional, and translational levels. Bacterial pathogens have a variety of mechanisms to adapt their membrane in response to transmission between environment, vector, and human host. Using a well-characterized model of lipid A diversification (Francisella), we demonstrate temperature-regulated membrane remodeling directed by multiple alleles of the lipid A-modifying N-acyltransferase enzyme, LpxD. Structural analysis of the lipid A at environmental and host temperatures revealed that the LpxD1 enzyme added a 3-OH C18 acyl group at 37 °C (host), whereas the LpxD2 enzyme added a 3-OH C16 acyl group at 18 °C (environment). Mutational analysis of either of the individual Francisella lpxD genes altered outer membrane (OM) permeability, antimicrobial peptide, and antibiotic susceptibility, whereas only the lpxD1-null mutant was attenuated in mice and subsequently exhibited protection against a lethal WT challenge. Additionally, growth-temperature analysis revealed transcriptional control of the lpxD genes and posttranslational control of the LpxD1 and LpxD2 enzymatic activities. These results suggest a direct mechanism for LPS/lipid A-level modifications resulting in alterations of membrane fluidity, as well as integrity and may represent a general paradigm for bacterial membrane adaptation and virulence-state adaptation.