Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence

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ABSTRACT

 

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FIG 2

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.

 

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. interrogans, m/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 (35, 44), 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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RESULTS

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 (33, 45). 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).

 

FIG 4

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.

 

 

FIG 5

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.

 

 

FIG 6

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.

 

FIG 7

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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DISCUSSION

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 (22, 47). 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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SUPPLEMENTARY MATERIAL

Supplemental material:

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ACKNOWLEDGMENTS

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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FOOTNOTES

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

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REFERENCES

1. Ko AI, Goarant C, Picardeau M. 2009. Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol 7:736–747. doi:10.1038/nrmicro2208. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
2. Hagen JE, Costa F, Kane M, Calcagno J, Ko AI. 2013. Global morbidity and mortality of leptospirosis: a systematic review, p 66 Abstr Sci Meet Int Leptospirosis Soc, Fukuoka, Japan. [Google Scholar]
3. Goswami RP, Basu A, Tripathi SK, Chakrabarti S, Chattopadhyay I. 2014. Predictors of mortality in leptospirosis: an observational study from two hospitals in Kolkata, eastern India. Trans R Soc Trop Med Hyg 108:791–796. doi:10.1093/trstmh/tru144. [PubMed] [CrossRef] [Google Scholar]
4. Faine S, Adler B, Bolin C, Perolat P. 1999. Leptospira and leptospirosis, p 30–32. MediSci, Melbourne, Australia. [Google Scholar]
5. Adler B, de la Peña Moctezuma A. 2010. Leptospira and leptospirosis. Vet Microbiol 140:287–296. doi:10.1016/j.vetmic.2009.03.012. [PubMed] [CrossRef] [Google Scholar]
6. Firth C, Bhat M, Firth MA, Williams SH, Frye MJ, Simmonds P, Conte JM, Ng J, Garcia J, Bhuva NP, Lee B, Che X, Quan PL, Lipkin WI. 2014. Detection of zoonotic pathogens and characterization of novel viruses carried by commensal Rattus norvegicus in New York City. mBio 5:e01933–01914. doi:10.1128/mBio.01933-14. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
7. Trueba G, Zapata S, Madrid K, Cullen P, Haake D. 2004. Cell aggregation: a mechanism of pathogenic Leptospira to survive in fresh water. Int Microbiol 7:35–40. [PubMed] [Google Scholar]
8. Barragan VA, Mejia ME, Trávez A, Zapata S, Hartskeerl RA, Haake DA, Trueba GA. 2011. Interactions of Leptospira with environmental bacteria from surface water. Curr Microbiol 62:1802–1806. doi:10.1007/s00284-011-9931-3. [PubMed] [CrossRef] [Google Scholar]
9. Ishii S, Sadowsky MJ. 2008. Escherichia coli in the environment: implications for water quality and human health. Microbes Environ 23:101–108. doi:10.1264/jsme2.23.101. [PubMed] [CrossRef] [Google Scholar]
10. Bleasdale B, Lott PJ, Jagannathan A, Stevens MP, Birtles RJ, Wigley P. 2009. The Salmonella pathogenicity island 2-encoded type III secretion system is essential for the survival of Salmonella enterica serovar Typhimurium in free-living amoebae. Appl Environ Microbiol 75:1793–1795. doi:10.1128/AEM.02033-08. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
11. Eisen RJ, Petersen JM, Higgins CL, Wong D, Levy CE, Mead PS, Schriefer ME, Griffith KS, Gage KL, Beard CB. 2008. Persistence of Yersinia pestis in soil under natural conditions. Emerg Infect Dis 14:941–943. doi:10.3201/eid1406.080029. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
12. Vezzulli L, Pruzzo C, Huq A, Colwell RR. 2010. Environmental reservoirs of Vibrio cholerae and their role in cholera. Environ Microbiol Rep 2:27–33. doi:10.1111/j.1758-2229.2009.00128.x. [PubMed] [CrossRef] [Google Scholar]
13. Lyczak JB, Cannon CL, Pier GB. 2000. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2:1051–1060. doi:10.1016/S1286-4579(00)01259-4. [PubMed] [CrossRef] [Google Scholar]
14. Nowicki EM, O'Brien JP, Brodbelt JS, Trent MS. 2014. Characterization of Pseudomonas aeruginosa LpxT reveals dual positional lipid A kinase activity and co-ordinated control of outer membrane modification. Mol Microbiol 94:728–741. doi:10.1111/mmi.12796. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
15. Li Y, Powell DA, Shaffer SA, Rasko DA, Pelletier MR, Leszyk JD, Scott AJ, Masoudi A, Goodlett DR, Wang X, Raetz CR, Ernst RK. 2012. LPS remodeling is an evolved survival strategy for bacteria. Proc Natl Acad Sci U S A 109:8716–8721. doi:10.1073/pnas.1202908109. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
16. Paciello I, Silipo A, Lembo-Fazio L, Curcurù L, Zumsteg A, Noël G, Ciancarella V, Sturiale L, Molinaro A, Bernardini ML. 2013. Intracellular Shigella remodels its LPS to dampen the innate immune recognition and evade inflammasome activation. Proc Natl Acad Sci U S A 110:E4345–E4354. doi:10.1073/pnas.1303641110. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
17. Reinés M, Llobet E, Dahlström KM, Pérez-Gutiérrez C, Llompart CM, Torrecabota N, Salminen TA, Bengoechea JA. 2012. Deciphering the acylation pattern of Yersinia enterocolitica lipid A. PLoS Pathog 8:e1002978. doi:10.1371/journal.ppat.1002978. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
18. Bengoechea JA, Brandenburg K, Arraiza MD, Seydel U, Skurnik M, Moriyón I. 2003. Pathogenic Yersinia enterocolitica strains increase the outer membrane permeability in response to environmental stimuli by modulating lipopolysaccharide fluidity and lipid A structure. Infect Immun 71:2014–2021. doi:10.1128/IAI.71.4.2014-2021.2003. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
19. Merino S, Camprubí S, Tomás JM. 1992. Effect of growth temperature on outer membrane components and virulence of Aeromonas hydrophila strains of serotype O:34. Infect Immun 60:4343–4349. [PMC free article] [PubMed] [Google Scholar]
20. Kawahara K, Tsukano H, Watanabe H, Lindner B, Matsuura M. 2002. Modification of the structure and activity of lipid A in Yersinia pestis lipopolysaccharide by growth temperature. Infect Immun 70:4092–4098. doi:10.1128/IAI.70.8.4092-4098.2002. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
21. Phillips NJ, Adin DM, Stabb EV, McFall-Ngai MJ, Apicella MA, Gibson BW. 2011. The lipid A from Vibrio fischeri lipopolysaccharide: a unique structure bearing a phosphoglycerol moiety. J Biol Chem 286:21203–21219. doi:10.1074/jbc.M111.239475. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
22. Sinensky M. 1974. Homeoviscous adaptation—a homeostatic process that regulates the viscosity of membrane lipids in Escherichia coli. Proc Natl Acad Sci U S A 71:522–525. doi:10.1073/pnas.71.2.522. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
23. Isogai E, Isogai H, Kurebayashi Y, Ito N. 1986. Biological activities of leptospiral lipopolysaccharide. Zentralbl Bakteriol Mikrobiol Hyg A 261:53–64. [PubMed] [Google Scholar]
24. Isogai E, Kitagawa H, Isogai H, Matsuzawa T, Shimizu T, Yanagihara Y, Katami K. 1989. Effects of leptospiral lipopolysaccharide on rabbit platelets. Zentralbl Bakteriol 271:186–196. doi:10.1016/S0934-8840(89)80072-6. [PubMed] [CrossRef] [Google Scholar]
25. Priya CG, Rathinam SR, Muthukkaruppan V. 2008. Evidence for endotoxin as a causative factor for leptospiral uveitis in humans. Invest Ophthalmol Vis Sci 49:5419–5424. doi:10.1167/iovs.08-2174. [PubMed] [CrossRef] [Google Scholar]
26. Segers RP, De Nijs A, van Kooten PJ, Gaastra W, van der Zeijst BA. 1990. The use of immunodeficient male (CBA/N x BALB/c) F1 mice to produce monoclonal antibodies directed to proteins of Leptospira interrogans rather than to immunodominant lipopolysaccharides. Hybridoma 9:275–283. doi:10.1089/hyb.1990.9.275. [PubMed] [CrossRef] [Google Scholar]
27. Cinco M, Delneri D, Banfi E. 1992. Immunodominant antigens recognized by the human immune response to infection by organisms of the species Leptospira interrogans serogroup Australis. FEMS Microbiol Immunol 4:287–297. [PubMed] [Google Scholar]
28. Werts C, Tapping RI, Mathison JC, Chuang TH, Kravchenko V, Saint Girons I, Haake DA, Godowski PJ, Hayashi F, Ozinsky A, Underhill DM, Kirschning CJ, Wagner H, Aderem A, Tobias PS, Ulevitch RJ. 2001. Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism. Nat Immunol 2:346–352. doi:10.1038/86354. [PubMed] [CrossRef] [Google Scholar]
29. Raddi G, Morado DR, Yan J, Haake DA, Yang XF, Liu J. 2012. Three-dimensional structures of pathogenic and saprophytic Leptospira species revealed by cryo-electron tomography. J Bacteriol 194:1299–1306. doi:10.1128/JB.06474-11. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
30. Murray GL, Srikram A, Henry R, Hartskeerl RA, Sermswan RW, Adler B. 2010. Mutations affecting Leptospira interrogans lipopolysaccharide attenuate virulence. Mol Microbiol 78:701–709. doi:10.1111/j.1365-2958.2010.07360.x. [PubMed] [CrossRef] [Google Scholar]
31. Marcsisin RA, Bartpho T, Bulach DM, Srikram A, Sermswan RW, Adler B, Murray GL. 2013. Use of a high-throughput screen to identify Leptospira mutants unable to colonize the carrier host or cause disease in the acute model of infection. J Med Microbiol 62:1601–1608. doi:10.1099/jmm.0.058586-0. [PubMed] [CrossRef] [Google Scholar]
32. Que-Gewirth NLS, Ribeiro AA, Kalb SR, Cotter RJ, Bulach DM, Adler B, Girons IS, Werts C, Raetz CRH. 2004. A methylated phosphate group and four amide-linked acyl chains in Leptospira interrogans lipid A: the membrane anchor of an unusual lipopolysaccharide that activates TLR2. J Biol Chem 279:25420–25429. doi:10.1074/jbc.M400598200. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
33. Bourhy P, Louvel H, Saint Girons I, Picardeau M. 2005. Random insertional mutagenesis of Leptospira interrogans, the agent of leptospirosis, using a mariner transposon. J Bacteriol 187:3255–3258. doi:10.1128/JB.187.9.3255-3258.2005. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
34. Slamti L, de Pedro MA, Guichet E, Picardeau M. 2011. Deciphering morphological determinants of the helix-shaped Leptospira. J Bacteriol 193:6266–6275. doi:10.1128/JB.05695-11. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
35. Eshghi A, Becam J, Lambert A, Sismeiro O, Dillies MA, Jagla B, Wunder EA, Ko AI, Coppee JY, Goarant C, Picardeau M. 2014. A putative regulatory genetic locus modulates virulence in the pathogen Leptospira interrogans. Infect Immun 82:2542–2552. doi:10.1128/IAI.01803-14. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
36. Eshghi A, Lourdault K, Murray GL, Bartpho T, Sermswan RW, Picardeau M, Adler B, Snarr B, Zuerner RL, Cameron CE. 2012. Leptospira interrogans catalase is required for resistance to H₂O₂and for virulence. Infect Immun 80:3892–3899. doi:10.1128/IAI.00466-12. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
37. Lourdault K, Aviat F, Picardeau M. 2009. Use of quantitative real-time PCR for studying the dissemination of Leptospira interrogans in the guinea pig infection model of leptospirosis. J Med Microbiol 58:648–655. doi:10.1099/jmm.0.008169-0. [PubMed] [CrossRef] [Google Scholar]
38. Lo M, Bulach DM, Powell DR, Haake DA, Matsunaga J, Paustian ML, Zuerner RL, Adler B. 2006. Effects of temperature on gene expression patterns in Leptospira interrogans serovar Lai as assessed by whole-genome microarrays. Infect Immun 74:5848–5859. doi:10.1128/IAI.00755-06. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
39. Eshghi A, Pappalardo E, Hester S, Thomas B, Pretre G, Picardeau M. 2015. The pathogenic Leptospira interrogans exoproteins are primarily involved in heterotrophic processes. Infect Immun 83:3061–3073. doi:10.1128/IAI.00427-15. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
40. Henderson JC, O'Brien JP, Brodbelt JS, Trent MS. 2013. Isolation and chemical characterization of lipid A from gram-negative bacteria. J Vis Exp 79:e50623. doi:10.3791/50623. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
41. Raetz CR, Purcell S, Meyer MV, Qureshi N, Takayama K. 1985. Isolation and characterization of eight lipid A precursors from a 3-deoxy-d-manno-octylosonic acid-deficient mutant of Salmonella typhimurium. J Biol Chem 260:16080–16088. [PubMed] [Google Scholar]
42. Caroff M, Deprun C, Karibian D, Szabó L. 1991. Analysis of unmodified endotoxin preparations by 252Cf plasma desorption mass spectrometry. Determination of molecular masses of the constituent native lipopolysaccharides. J Biol Chem 266:18543–18549. [PubMed] [Google Scholar]
43. Bligh EG, Dyer WJ. 1959. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917. doi:10.1139/o59-099. [PubMed] [CrossRef] [Google Scholar]
44. King AM, Pretre G, Bartpho T, Sermswan RW, Toma C, Suzuki T, Eshghi A, Picardeau M, Adler B, Murray GL. 2014. High-temperature protein G is an essential virulence factor of Leptospira interrogans. Infect Immun 82:1123–1131. doi:10.1128/IAI.01546-13. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
45. Murray GL, Morel V, Cerqueira GM. 2009. Genome-wide transposon mutagenesis in pathogenic Leptospira spp. Infect Immun 77:810–816. doi:10.1128/IAI.01293-08. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
46. Pilizota T, Shaevitz JW. 2013. Plasmolysis and cell shape depend on solute outer membrane permeability during hyperosmotic shock in E. coli. Biophys J 104:2733–2742. doi:10.1016/j.bpj.2013.05.011. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
47. Cronan JE, Gelmann EP. 1975. Physical properties of membrane lipids: biological relevance and regulation. Bacteriol Rev 39:232–256. [PMC free article] [PubMed] [Google Scholar]

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