Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence

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Leptospirosis is an emerging disease with an annual occurrence of over 1 million human cases worldwide. Pathogenic Leptospira bacteria are maintained in zoonotic cycles involving a diverse array of mammals, with the capacity to survive outside the host in aquatic environments. Survival in the diverse environments encountered by Leptospira likely requires various adaptive mechanisms. Little is known about Leptospira outer membrane modification systems, which may contribute to the capacity of these bacteria to successfully inhabit and colonize diverse environments and animal hosts. Leptospira bacteria carry two genes annotated as UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase genes (la0512 and la4326 [lpxD1 and lpxD2]) that in other bacteria are involved in the early steps of biosynthesis of lipid A, the membrane lipid anchor of lipopolysaccharide. Inactivation of only one of these genes, la0512/lpxD1, imparted sensitivity to the host physiological temperature (37°C) and rendered the bacteria avirulent in an animal infection model. Polymyxin B sensitivity assays revealed compromised outer membrane integrity in the lpxD1 mutant at host physiological temperature, but structural analysis of lipid A in the mutant revealed only minor changes in the lipid A moiety compared to that found in the wild-type strain. In accordance with this, an in trans complementation restored the phenotypes to a level comparable to that of the wild-type strain. These results suggest that the gene annotated as lpxD1 in Leptospira interrogans plays an important role in temperature adaptation and virulence in the animal infection model.


Members of the genus Leptospira are spirochete bacteria encompassing saprophytic and pathogenic species and are considered to be the most widespread zoonotic bacteria worldwide (1). Leptospira is the etiological agent of the disease leptospirosis, which in severe manifestations leads to hemorrhage in the lungs, meningitis, and liver and/or kidney failure (1). Leptospirosis is an emerging disease, and the worldwide annual occurrence is estimated to be over 1 million human cases, with a 5 to 20% mortality rate (23). Leptospira cannot breach the host epidermal lining, and transmission requires direct contact of the bacteria with cuts or abrasions in the skin (45). Rats and other rodent species serve as reservoir hosts for Leptospira, which colonizes the urinary systems of these animals (6). Leptospira is shed back into the environment through the urine of reservoir hosts and can persist in freshwater and soil until direct contact with an animal recommences an infection cycle (78).

A prominent Leptospira feature is the ability to proliferate under significantly different environmental conditions. Other Gram-negative bacterial pathogens with this ability include species of the genera Escherichia (9), Salmonella (10), Yersinia (11), Vibrio (12), and Pseudomonas (13). The capacity of bacteria to adapt to disparate environments is likely imparted by numerous evolved strategies that probably include modification of outer membrane macromolecules (14,–17). Outer membrane fluidity and permeability are partly modulated by hydrophobic acyl chains of lipid A regions of lipopolysaccharide (LPS) (18). Environmental fluctuations, such as temperature, alter outer membrane fluidity (19), and bacteria counter this by altering acyl chain lengths or the number of acyl groups added to the lipid A moiety and/or by the modification of acyl chain saturation to maintain outer membrane integrity (20,–22).

Measurements of the toxicity of Leptospira interrogans LPS suggest that while the molecule is less toxic than Escherichia coli LPS (23), L. interrogans LPS is toxic to a variety of cells (24) and tissues encountered by the pathogen during the course of animal infection (25). L. interrogans LPS is also an immunodominant molecule (2627) and is unique in that it is recognized by Toll-like receptor 2 (TLR2) and not TLR4 in human cells (LPS from the majority of other Gram-negative bacteria is recognized by TLR4) (28). Pathogenic Leptospira strains demonstrate more abundant and longer LPS than the saprophyte Leptospira biflexa (29), and mutations affecting the native biosynthesis of LPS affect both virulence in hamsters (30) and colonization of target organs in the mouse model (31).

Acylation of lipid A has been shown to be crucial for the fitness of bacteria outside and within the host (152021). The L. interrogans genome encodes homologues of the enzymes required for lipid A biosynthesis, and this biosynthetic process has been previously proposed in L. interrogans (32). Structural analyses of L. interrogans serovars Pomona and Icterohaemorrhagiae (strain Verdun) lipid A have been performed, revealing identical structures composed of a 2,3-diamino-2,3-dideoxy-d-glucopyranose disaccharide with four amide-linked acyl groups composed of R-3-hydroxylaurate at positions 3 and 3′ and R-3-hydroxypalmitate at positions 2 and 2′ (32). Two secondary unsaturated acyl chains are ester linked to the 2′ and 3′ hydroxy-acyl groups to produce hexa-acylated lipid A as the major species (32). Previous structural analysis of L. interrogans lipid A suggested that the C-2 and C-2′ amine groups are acylated with 16 carbon length hydroxy-acyl groups (32), which suggests that the L. interrogans LpxD enzyme is selective for 16-carbon 3-hydroxy-acyl chains. The L. interrogans serovar Manilae examined in this report has two genes (la0512 and la4326) that display homology to lpxD in other Gram-negative bacteria. The present study aimed to characterize pathogenic L. interrogans serovar Manilae lpxD homologues in the context of outer membrane integrity conferring temperature adaptation and virulence in an animal infection model.

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Leptospira strains and culture.

Leptospira interrogans serovar Manilae strain L495, the la0512 (lpxD1) and la4326 (lpxD2) mutants, and the la0512 mutant complemented with la0512 (the lpxD1 complemented mutant) were maintained in Ellinghausen and McCullough as modified by Johnson and Harris (EMJH) growth medium at 30°C with agitation.

Insertion mutagenesis and complementation.

Insertion inactivation in L. interrogans has been described previously (33). The insertion sites within la0512 (lpxD1 mutant) and la04326 (lpxD2 mutant) were identified by semirandom PCR, followed by DNA sequencing. The insertion was further confirmed via PCR using primers flanking the insertion sites. For complementation, the lpxD1 mutant was PCR amplified using primers lpxD1F (5′-GGGAATTCCATATGAAAGCCAAAAATTTAGCG-3′) and lpxD1R (5′-CGGCTCGAGATCCAATTCAACCTG-3′), which incorporated restriction digestion sites for NdeI and XhoI, respectively (underlined bases). The lpxD1 coding sequence was then digested with NdeI and XhoHI, purified, and inserted into the same restriction sites of pCRPromFlgB (34) to generate a transcriptional fusion between the gene and the Borrelia burgdorferi flgB promoter. DNA fragments containing the transcriptional fusions were released by KpnI and XhoI digestions and cloned into the corresponding sites of pAL614 (a generous gift from Gerald Murray, Monash University). The lpxD1 complementation construct was introduced by conjugation in the Manilae lpxD1 mutant strain as previously described (35), and complementation of the lpxD1 mutant strain was confirmed by using primers that PCR amplified a region of the spectinomycin resistance cassette and primers lpxD1F and lpxD1R, using genomic DNA as the template.

Growth rate measurements, susceptibility assays, and morphology determination.

To determine whether mutant, complemented, and wild-type (wt) Leptospira strains were affected in growth in vitro, bacteria were enumerated using a Petroff-Hausser counting chamber (Hausser Scientific Company, Horsham, PA, USA) under dark-field microscopy. A total of 2,000 bacteria of each strain were used to inoculate 9 ml EMJH medium in triplicate. Growth was monitored on a daily basis at 30°C and 37°C by counting bacteria using a Petroff-Hausser counting chamber under dark-field microscopy. For enumerating strains on a daily basis, equal-volume aliquots of samples were pooled and counted in duplicate. Growth experiments were performed 4 times with similar trends; a representative result from a single experiment is displayed in Fig. 1.





Inactivation of Leptospira lpxD1 results in temperature-sensitive growth. The Leptospira parent wt, lpxD1 mutant, lpxD2 mutant, and lpxD1 complemented mutant strains were used to compare temperature sensitivity. (A) The strains were cultured in triplicate at 30°C, and cell density was measured for each strain by pooling replicates and counting cells daily using Petroff-Hauser counting chambers under dark-field microscopy. (B) The strains were cultured at 37°C, and cell densities were determined as for 30°C cultures. (C) Temperature sensitivity was also determined by exposing strains at a density of 5 × 106/ml to the indicated temperatures for 72 h. Cell viability was subsequently measured using an alamarBlue assay, where pink represents viable cells, blue represents nonviable cells, and intermediate between pink and blue represents reduced viability.

Temperature sensitivity was measured by adding 200 μl of bacteria at a concentration of 5 × 106 bacteria/ml in EMJH medium to 96-well culture plates in triplicate. The plates were sealed to prevent evaporation and incubated at 23°C, 30°C, or 37°C for 72 h. To measure cell viability, bacterial cultures were transferred to a single 96-well culture plate, 20 μl of alamarBlue (Life Technologies SAS, Saint Aubin, Île-de-France, France) was added to the bacteria, and the plates were incubated for 24 h at 30°C. Temperature sensitivity experiments were performed twice with similar results, and a representative result from a single experiment is displayed in Fig. 1. For polymyxin B (Sigma, Saint Louis, MO, USA), sensitivity assay experiments were performed as described for the temperature sensitivity assays with the following modifications. Polymyxin B was added to 96-well plates in the range of 20 μg/ml to 0.02 μg/ml, using 2-fold serial dilutions. Bacteria were added to a final volume of 200 μl and a final concentration of 5 × 105 bacteria/ml. The plates were sealed to prevent evaporation and incubated at 30°C or 37°C for 24 h, 20 μl of alamarBlue was subsequently added per well, and the plates were sealed and incubated at 30°C for 48 h to measure cell viability. For these experiments, the starting bacterial concentration was 5 × 105 (as opposed to 5 × 106 for the temperature sensitivity assays described above) to ensure lpxD1 mutant viability at 37°C, as the viability of the mutant rapidly declines with increasing bacterial concentration at 37°C. Polymyxin B experiments were performed two times with comparable results, and a representative result from a single experiment is displayed in Fig. 2.



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Reduced outer membrane integrity in the lpxD1 mutant at 37°C. The relative outer membrane permeability of the Leptospira parent wt, lpxD1 mutant, lpxD2 mutant, and lpxD1 complemented mutant strains was tested by measuring MICs to the cationic compound polymyxin B. Bacteria were exposed to polymyxin B in the indicated concentration range (using successive 2-fold dilutions) for 72 h at the indicated temperatures. Cell viability was measured using the alamarBlue assay; viable bacteria are represented in the pink wells and nonviable bacteria in the blue wells.

Morphological changes were measured by first growing bacterial strains at 23°C, 30°C, or 37°C to a density of 5 × 106 bacteria/ml in triplicate. Ten-microliter aliquots of bacteria were viewed by dark-field microscopy at ×200 magnification, and images were captured using Olympus CellSens Dimensions version 1.7.1 (Olympus, Rungis, Île-de-France, France). Twenty bacteria were measured lengthwise (using the CellSens Dimensions version 1.7.1 measuring tool) per replicate for a total of 60 bacteria per strain from each temperature. Statistical analysis was performed using two-way analysis of variance (ANOVA).

RNA extraction and RT-qPCR.

Leptospira strains were cultured in triplicate at 30°C in EMJH medium to a density of 1 × 108 bacteria/ml, and a total of 1010 bacteria of each strain from each replicate were used for RNA extraction and reverse transcription–quantitative real-time PCR (RT-qPCR), as previously described (35,–38). The following modifications were implemented for RT-qPCR: the primers used to quantify the genes were lpxD1f (5′-ATCCGAACGTTGTCATTGAA) and lpxD1r (5′-GATCACCGTATTCGCATGAA) to quantify lpxD1 mutant transcripts and lpxD2f (5′-TCATCCTTCTGCAAAGTTGG) and lpxD2r (5′-AACGCCGTCTTCCAAATAAG) to quantify lpxD2 mutant transcripts. Statistical analysis was performed using both biological replicates (n = 3) and RT-qPCR technical replicates (n = 3) with an unpaired Student t test, comparing strains individually.

LPS purification and immunoblot analyses.

Leptospira strains were cultured at 37°C in EMJH medium to a density of 1 × 107 bacteria/ml and harvested via centrifugation at 9,000 × g for 10 min to obtain a total of 1010 cells of each strain. The LPS was purified as previously described (30) with the following modifications. Immediately after harvesting, the Leptospira pellets were resuspended in 1× phosphate-buffered saline (PBS)-0.1% SDS to a concentration of 109 bacteria/ml and sonicated for 45 s at 20 W. Proteinase K was added to a final concentration of 30 μg/ml, and samples were incubated at room temperature for 24 h on a RotoFlex Tube Rotator (Argos Technologies, Elgin, IL, USA). Samples were subsequently mixed with 4× Laemmli protein sample buffer (Bio-Rad, Marnes-la-Coquette, Île-de-France, France) and used for SDS-PAGE, silver staining, and immunoblot analysis with an equivalent volume of 1 × 107 bacteria per lane. Immunoblots were performed using L. interrogans serovar Manilae strain L495-positive guinea pig sera, which were obtained as previously described (39), at a 1:100 dilution in 1× PBS-5% (wt/vol) skim milk-0.1% Tween 20 (PBSMT). The guinea pig sera were detected with horseradish peroxidase-conjugated goat anti-guinea pig immunoglobulins as previously described (39). These experiments were performed 3 times with similar results, and the result from a single experiment is displayed in Fig. 3.



Inactivation of lpxD1 results in lowered immunoreactivity to LPS. Crude LPS extracts of bacteria grown at 37°C were applied to SDS-PAGE and used in immunoblot experiments with sera from guinea pigs infected with wild-type Manilae. (A) Silver-stained SDS-PAGE of crude LPS from the indicated strains. (B) Immunoblot displaying IgG reactivities of Leptospira-positive sera against crude LPS from the indicated strains.

Isolation and matrix-assisted laser desorption ionization (MALDI)–MS analysis of lipid A.

Leptospira strains were grown in EMJH medium at 30°C or 37°C to a density of 3 × 107 bacteria/ml, and a total of 3 × 1010 bacteria of each strain from each temperature were used for lipid A isolation. Lipid A isolation and mass spectrometry (MS) were performed as previously described (40) with the following modifications. Cells were harvested by centrifugation, washed with PBS, and stored at −20°C until lipid A extraction. Previously described modifications to the methods of Caroff et al. and Raetz et al. were used to chemically isolate lipopolysaccharide, where isolated material was treated by boiling-mild-acid hydrolysis for 45 min to liberate lipid A from attached polysaccharide (40,–42). The method of Bligh and Dyer was used to extract lipid A after mild-acid hydrolysis (43). The isolated lipid A was dried under nitrogen and stored at −20°C until further use.

Lipid A extracts were further purified over a DEAE column to improve the quality of spectra obtained by MALDI-MS. Briefly, dried lipid A was suspended in 2:3:1 (vol/vol/vol) chlorofom-methanol-water (CMW) and applied to a 1.5-ml preequilibrated DEAE column. The column was washed with 20 column volumes of 2:3:1 CMW, and lipid A species were eluted stepwise with 5 column volumes of 2:3:1 CMW-ammonium acetate at 60 mM, 120 mM, 240 mM, or 480 mM ammonium acetate. An additional two-phase Bligh-Dyer extraction was performed on the eluate to remove the ammonium acetate. The isolated lipid A was dried under nitrogen and stored in small conical glass vials at −20°C until MALDI-MS analysis. Lipid A was resuspended in 25 μl chloroform-methanol (4:1). An empirically determined amount of lipid A, varied per sample, was mixed with 0.5 μl of matrix (saturated 6-aza-2-thiothymine in 50% acetonitrile-saturated tribasic ammonium citrate [20:1 {vol/vol}]) and spotted on a 100-well MALDI plate. An AB Sciex Voyager was used to collect MALDI-time of flight (TOF)–MS data in the negative reflectron mode. Consistent with previous reports on L. interrogansm/z peaks corresponding to lipid A species were present in most of the 60 mM ammonium acetate fraction spectra (32). The proposed absence of the 4′-phosphate group and a methylated 1-phosphate contribute to the early elution of lipid A from L. interrogans (32). No peaks corresponding to lipid A were observed in the later 120, 240, and 480 mM ammonium acetate fractions. The spectra obtained represent the average of >300 shots.

Gerbil infection and bacterial burden in target organs.

An initial virulence experiment was performed in gerbils with the Manilae wt strain and the lpxD1 mutant and lpxD2 mutant strains. For these experiments, groups of 4 gerbils were challenged intraperitoneally with each of the above-mentioned strains using 104 bacteria per animal. The animals were monitored for 20 days and euthanized, when possible, to minimize animal suffering. A second infection experiment was performed as described above with the following modification: the lpxD1 complemented mutant strain was used in place of the lpxD2 mutant strain, and infections were performed at doses of 104 and 106 bacteria per animal in groups of 4 gerbils per bacterial dose.

Leptospira burdens in kidneys and liver were determined by qPCR, as previously described (3544), with the following modifications. The Leptospira Manilae wild-type, lpxD1 mutant, and lpxD1 complemented mutant strains were injected intraperitoneally into groups of 4 gerbils (104 bacteria per animal). Five days after the injection of bacteria, the animals were euthanized, and the kidneys and liver from each animal were harvested for culturing in EMJH medium and for qPCR. Calculations of the bacterial burden were performed to obtain the number of bacteria per 100 mg organ. An F test to compare variance (P < 0.0064) was used to compare the bacterial burdens in target organs.

Ethics statement.

The protocols for the animal experiments were prepared according to the guidelines of the Animal Care and Use Committee of Institut Pasteur of Paris, and the present study was approved by that committee (no. CETEA 2013-0019).

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lpxD1 inactivation reduces L. interrogans fitness at the host physiological temperature.

LpxD enzymes have been shown to contribute to bacterial temperature adaptation via modification of the acyl chain length of the lipid A region of lipopolysaccharide (15). To determine whether lpxD genes contained similar functionality in Leptospira, strains with Himar1 transposon insertions in the genes la0512 and la4326 (lpxD1 mutant and lpxD2 mutant, respectively) were obtained from a previously generated library of transposon insertion mutants (3345). The in vitro growth rates of the lpxD1 mutant (la0512) and lpxD2 mutant (la4326) strains were compared to the Manilae L495 parent strain (wt) at 30°C and 37°C (Fig. 1A and andB).B). These experiments showed comparable growth rates for both mutants and the wt at 30°C; however, at 37°C, reduced cell density was observed in the lpxD1 mutant relative to the wt and the lpxD2 mutant (Fig. 1B). To further test lpxD1 mutant temperature susceptibility, the mutant strains were cultured to a density of 5 × 106/ml (approaching the approximate density at which the lpxD1 mutant lost viability at 37°C) in growth medium and incubated at 23°C, 30°C, and 37°C for 72 h. Following the 72 h of incubation, bacterial viability was measured via alamarBlue viability assay, which demonstrated that the lpxD1 mutant was not viable at 37°C (Fig. 1C).

To validate these observations, the lpxD1 mutant was complemented with the native lpxD1 gene under the control of a constitutive promoter (technical limitations prevented complementation with the native lpxD1 promoter). The resulting strain (lpxD1 complemented mutant) displayed a surprisingly higher growth rate at 37°C and reached a higher density at both 30°C and 37°C than the mutant and wt strains (Fig. 1A and andB).B). RT-qPCR was used to measure the relative lpxD1 transcription in the strains, demonstrating a >2.5-fold increase in lpxD1 transcription in the lpxD1 complemented mutant compared to the wt (see Fig. S1 in the supplemental material).

Increased outer membrane permeability in the lpxD1 mutant.

Outer membrane integrity was assessed using polymyxin B assays conducted at 23°C, 30°C, and 37°C, and the viability of the bacteria was determined using alamarBlue assays (Fig. 2A). Interestingly, the lpxD2 mutant displayed 2-fold-higher resistance to polymyxin B, and this resistance was independent of the tested temperatures (Fig. 2A). In contrast, the lpxD1 mutant exhibited 2-fold-higher sensitivity to polymyxin B at 37°C, but not at 30°C or 23°C, than the other strains (Fig. 2A). The wt and lpxD1 complemented mutant strains displayed comparable polymyxin B susceptibilities (Fig. 2A).

Reduced outer membrane integrity can lead to osmotic stress, resulting in reduced cell size (46). To assess whether strains displayed morphological differences, the cell lengths of the mutant, complemented, and wt strains were measured using dark-field microscopy (see Fig. S2 in the supplemental material). These analyses revealed that at 37°C, the cell length of the lpxD1 mutant was reduced by 16% (1.5 μm on average) compared to the other strains (see Fig. S2 in the supplemental material).

lpxD1 is required for leptospiral kidney and liver colonization and for fatal leptospirosis in gerbils.

Immunoblot analysis using Leptospira-positive sera and total LPS from the mutant, complemented, and wt strains grown at 37°C demonstrated reduced reactivity with LPS obtained from the lpxD1 mutant (Fig. 3), providing indirect evidence of lpxD1 function in the host. To ascertain whether lpxD1 function was also required for survival of Leptospira in the host, the lpxD1 mutant, lpxD2 mutant, and wt strains were used in virulence experiments in the gerbil infection model (Fig. 4A). These experiments revealed that when animals were injected intraperitoneally with 104 bacteria per gerbil, those infected with the lpxD1 mutant strain survived the 20-day infection experiment (Fig. 4A) without visible signs of morbidity, whereas animals infected with the lpxD2 mutant and those infected with the wt died by day 7. In separate experiments, the lpxD1 mutant, lpxD1 complemented mutant, and wt strains were injected into the intraperitoneal cavities of gerbils at doses of 104 or 106 (Fig. 4B). Animals infected with the lpxD1 mutant strain at a dose of 104 or 106 per animal remained asymptomatic for the duration of the 20-day experiment, whereas animals infected with the wt or lpxD1 complemented mutant strain died at days 5 and 7 postinfection when injected with doses of 106 or 104 bacteria, respectively (Fig. 4B).



The lpxD1 mutant does not colonize target organs in the gerbil infection model. The Leptospira parent wt, lpxD1 mutant, lpxD2 mutant, and lpxD1 complemented mutant strains were used to assess virulence in gerbils. (A) Groups of 4 gerbils were inoculated with the indicated numbers of bacteria of each strain intraperitoneally and monitored for 20 days. (B) Infection experiments were performed as described for panel A. (C) Groups of 4 gerbils were inoculated intraperitoneally with 104 bacteria of each indicated strain per animal. Five days postinoculation, the animals were euthanized and the livers and kidneys were taken for qPCR and in vitro culturing of bacteria in EMJH medium. a, culture positive from both liver and kidney; b, negative for culture growth from liver and kidney.

For analysis of bacterial burdens in the kidneys and liver, gerbils were injected intraperitoneally with 104 bacteria of the lpxD1 mutant, lpxD1 complemented mutant, or wt strain, and 5 days postinfection, the animals were sacrificed to obtain kidneys and livers for qPCR and for in vitro culturing. Quantitative real-time PCR led to the detection of lpxD1 mutant DNA in the kidneys and livers in an approximately 10-fold-lower quantity than the wt (Fig. 4C). To distinguish between viable bacteria that had established colonization and those that reached the target organs but died shortly after, the organs were also used for in vitro culturing. The wt and lpxD1 complemented mutant strains were culture positive in all of the kidneys and livers tested, but the lpxD1 mutant strain was negative for growth (data not shown).

Structural analyses of Leptospira lipid A.

MALDI-MS analysis of lipid A species isolated from L. interrogans serovar Manilae produced data that are similar to previously published reports on serovars Pomona and Icterohaemorrhagiae (32). The previously proposed structure of L. interrogans lipid A is hexa-acylated, contains a methylated 1-phosphate, and lacks a 4′-phosphate group (33) (Fig. 5). Consistent with this structure, fractionation of L. interrogans serovar Manilae lipid A using anion-exchange chromatography (DEAE) revealed early elution (60 mM ammonium acetate) of lipid A species, consistent with a net decrease in anionic character due to the absence of a 4′-phosphate group and the presence of a methylated 1-phosphate group. Unmodified hexa-acylated bis-phosphorylated lipid A typically elutes in the 240 mM ammonium acetate fraction during DEAE fractionation. Unique to our analysis of serovar Manilae is an overall reduction in the observed m/z relative to serovars Pomona and Icterohaemorrhagiae by m/z 2 (Fig. 6A) (37°C) or 4 (Fig. 7A) (30°C), consistent with the incorporation of fatty acids with 1 or 2 more degrees of unsaturation (Fig. 5). The location of the unsaturated bond cannot be determined by MALDI-MS.




Proposed chemical structures of the major lipid A species from L. interrogans. Shown are chemical representations of the predominant species of lipid A in serovars Pomona and Verdun, as proposed in a previous report (32), and serovar Manilae, the putative structure proposed in this report. The exact mass and the monoisotopic mass (m/z) for MS in the negative mode are displayed.




MALDI-MS analysis of DEAE fractionated lipid A from L. interrogans serovar Manilae grown at 37°C. Lipid A isolated from each indicated strain was fractionated over a DEAE anion-exchange column. Mass spectra were obtained using the 60 mM ammonium acetate DEAE fraction for lipid A isolated from the WT (A), lpxD1 mutant (B), lpxD1 complemented mutant (C), and lpxD2 mutant (D) strains of L. interrogans serovar Manilae. Each spectrum is the average of >300 laser pulses. Based on the relative percent signal intensity, the most abundant m/z peak for each isotopic cluster is labeled.



MALDI-MS analysis of DEAE-fractionated lipid A from L. interrogans serovar Manilae grown at 30°C. Lipid A isolated from each indicated strain was fractionated over a DEAE anion-exchange column. Mass spectra were obtained using the 60 mM ammonium acetate DEAE fraction for lipid A isolated from WT (A), lpxD1 mutant (B), lpxD1 complemented mutant (C), and lpxD2 mutant (D) L. interrogans serovar Manilae. Each spectrum is the average of >300 laser pulses. Based on the relative percent signal intensity, the most abundant m/z peak for each isotopic cluster is labeled.

In all spectra, the predominant, putative lipid A species peak is flanked by less intense peaks at m/z −28 or +28 (Fig. 6 and and7),7), consistent with a lipid A species containing acyl chains that vary in length by 2 less or 2 more carbons, respectively. These peaks are also observable in MS data from the previously characterized serovars Pomona and Icterohaemorrhagiae (32). In serovar Manilae, an even shorter acyl chain containing lipid A is present in the lpxD1 mutant, which varies from the predominant peak by m/z −56, corresponding to a reduction in acyl chain length of 4 carbons (Fig. 6B and and7B).7B). Complementation of the lpxD1 mutant with a wild-type copy of lpxD1 resulted in the disappearance of this peak at either temperature (30°C or 37°C), producing a spectrum similar to that of the wild type (Fig. 6C and and7C).7C). Analysis of lipid A from the lpxD2 mutant resulted in a spectrum very similar to that obtained for the wild type at 37°C (Fig. 6D). However, the MALDI-MS spectra obtained from lipid A purified from the lpxD2 mutant grown at 30°C consistently produced a spectrum lacking the major spectral peaks (Fig. 7D) at m/z 1,691, 1,719, and 1,747 observed in wild-type samples (Fig. 7A).

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Phenotypic characterization of the mutant and the lpxD1 complemented mutant strains demonstrated that lpxD1 is required for optimal bacterial growth and survival at temperatures corresponding to that found within the host (37°C). Furthermore, the temperature sensitivity of the lpxD1 mutant at 37°C was at least partially a result of increased outer membrane permeability, as demonstrated by polymyxin B susceptibility assays. We did not assess bacterial growth at temperatures lower than 23°C, as the Leptospira growth rate rapidly declines at lower temperatures in vitro. Therefore, we cannot exclude the importance of lpxD2 for bacterial viability at lower temperatures. The use of two different LpxD enzymes for temperature adaptation has been previously demonstrated in the Gram-negative bacterial species Francisella (15).

Consistent with the requirement for lpxD1 functionality at 37°C, the comparative immunoreactivity of whole LPS molecules from the wt, mutant, and complemented strains suggested that lpxD1 is functional during the infection process in animals. Furthermore, virulence experiments in animals indicated that lpxD1 was essential for the ability of Leptospira to colonize target organs and to cause fatal disease in gerbils. The lack of lpxD1 expression and functionality in the lpxD1 mutant likely rendered Leptospira ineffective in maintaining a stable outer membrane for proper functioning of the bacteria at the elevated temperatures found within the host. Reduced fitness within the host due to increased outer membrane permeability was also likely further compounded by increased susceptibility to host antimicrobial peptides.

MALDI-MS analysis of lipid A species from all Leptospira strains used in the present study demonstrated a somewhat heterogeneous mixture with respect to degrees of unsaturation and overall acyl chain length. The predominant species observed corresponds to the proposed structure in Fig. 5. Spectra from the lpxD1 mutant strain displayed an additional lower m/z peak suggestive of a lipid A moiety with shortened acyl chain length. The appearance of the lipid A species containing a smaller acyl chain in the lpxD1 mutant might suggest that the protein product of lpxD2 (if indeed functional) has a more relaxed acyl chain substrate specificity than that of lpxD1, allowing the incorporation of even smaller acyl chains into the final lipid A structure. Comparison of the major lipid A species for L. interrogans serovar Manilae showed that at lower temperatures the most predominant lipid A species at 30°C (m/z 1,719.7) contained one more degree of unsaturation relative to the predominant species at 37°C (m/z 1,722.0). The homeoviscous adaptation hypothesis for bacterial membranes predicts increased unsaturated fatty acid content at low temperatures, which promotes the increase in fluidity required for optimal bacterial membrane functionality (2247). Direct biochemical characterization of LpxD1 and LpxD2 will be required to determine the precise contribution of each enzyme to the observed lipid A species in this study. It is also possible that changes in fatty acid donor pools (the ratios of various acyl-ACPs) as a consequence of varying growth temperatures or the activity of secondary lipid A acyltransferases, LpxL or LpxM, might contribute to the observed changes in lipid A acyl chain length/unsaturation.

The results of the present study are in agreement with previous theories on bacterial mechanisms used for modulating outer membrane integrity for bacterial adaptation to new environments. Unlike other bacteria, such as Yersinia and Neisseria, the total number of L. interrogans lipid A acyl chains appeared to remain constant under the various temperatures tested. In the case of L. interrogans, the effects of acyl chain length and the level of acyl chain saturation on thermal acclimatization and virulence in the animal infection model remain unclear. These results allow us to extrapolate that L. interrogans likely utilizes lpxD1 function to maintain outer membrane integrity when transmitted from niche inanimate environments, such as soil and water, to the animal host.

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We thank Catherine Werts for discussions and critical reading of the manuscript.

This work was funded by the FUI 14 (Fonds Unique Interministériel) COVALEPT and BPIFrance. Azad Eshghi was funded by a Carnot grant of the Institut Carnot-Pasteur Maladies Infectieuses. Also acknowledged are NIH grants AI064184 and AI076322 (M.S.T.) and grant W911NF-12-1-0390 from the Army Research Office (M.S.T.)

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Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00897-15.

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Leptospira antigens

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