Two Distinct L-Lactate Dehydrogenases Play a Role in the Survival of Neisseria gonorrhoeae in Cervical Epithelial Cells

Abstract

L-lactate is an abundant metabolite in a number of niches in host organisms and represents an important carbon source for bacterial pathogens such as Neisseria gonorrhoeae. In this study, we describe an alternative, iron-sulfur cluster-containing L-lactate dehydrogenase (LutACB), that is distinct from the flavoprotein L-lactate dehydrogenase (LldD). Expression of lutACB was found to be positively regulated by iron, whereas lldD was more highly expressed under conditions of iron-limitation. The functional role of LutACB and LldD was reflected in in vitro studies of growth and in the survival of N gonorrhoeae in primary cervical epithelial cells.

L-lactate is an important carbon source for Neisseria gonorrhoeae. Here, we describe an alternative L-lactate dehydrogenase, LutACB, which it is positively regulated by iron, and demonstrate its role in lactate respiration and survival of Neisseria gonorrhoeae in cervical epithelial cells.

L-lactate is produced by host tissues and commensal organisms and is present at concentrations approximately 1 mM in human serum and over 6 mM within the female genital tract [1]. L-lactate is a potential carbon and energy source for pathogenic bacteria such as the host-adapted human pathogen Neisseria gonorrhoeae [2, 3]. It is established that N gonorrhoeae can respire using both L-lactate and D-lactate, because it possesses a L-lactate permease (LctP), a membrane bound L-lactate dehydrogenase (LldD), and two D-lactate dehydrogenases (LdhA and LdhD) [3, 4]. This L-lactate and D-lactate oxidation pathway is linked to respiratory electron transfer and adenosine triphosphate synthesis [4]. The lactate utilization pathway is a key virulence determinant in N gonorrhoeae, because its disruption results in reduced survival and pathogenesis. For example, loss of LctP decreases colonization of the vaginal tract in a murine infection model, and loss of ldhA and lldD results in decreased survival in primary human cervical epithelial (pex) cells [3, 4].

The L-lactate dehydrogenase, LldD, was originally thought to be the only enzyme involved in L-lactate oxidation. However, growth of an N gonorrhoeae ΔlldD mutant was not affected when L-lactate was the sole carbon source [4], suggesting the existence of an alternative lactate utilization (Lut) pathway. Some bacteria have a distinct Lut pathway encoded by lutA-lutC-lutB [5]. In Campylobacter jejuni, LutA is identified as the main substrate-binding site, whereas LutB and LutC are associated with electron transport [6]. In this study, we investigated homologs of LutACB in N gonorrhoeae strain 1291, which are encoded by genes NGO0904-0906. Although this gene cluster was previously identified as an operon that is positively regulated by iron and the ferric iron-uptake regulator (Fur) in N gonorrhoeae, it was annotated as encoding a glycolate oxidase [7]. Therefore, we sought to confirm the function of LutACB as an alternative L-lactate dehydrogenase in N gonorrhoeae, to investigate the effect of iron on lutACB expression, as well as to define the role of this enzyme in the intracellular survival of N gonorrhoeae.

METHODS

Bacterial Strains and Growth Conditions

Neisseria gonorrhoeae strain 1291 and isogenic mutant strains were routinely cultured as described in [4]. All gonococci in this study were piliated (P⁺) and expressed Opa (Opa⁺). When required, medium was supplemented with chloramphenicol (2 µg/mL), kanamycin (100 µg/mL), or spectinomycin (100 µg/mL).

Deoxyribonucleic Acid Manipulations and Genetic Techniques

Neisseria gonorrhoeae mutants and complemented mutants were constructed exactly as previously described [4]. The lutACB (NGO0904-0906) and lctP (NGO_01449) were replaced with a chloramphenicol or kanamycin resistance cassette, respectively. The resulting chloramphenicol-resistant mutants were designated as Δlut or ΔΔlldD/lut, respectively, and the kanamycin-resistant mutant was designated ΔlctP. Spectinomycin-resistant complemented strains were designated as 1291Δlut::lut or 1291ΔΔlldD/lut::lut. All mutants were confirmed by deoxyribonucleic acid (DNA) sequencing at the Australian Equine Genome Research Facility (The University of Queensland, Australia). Primers used are described in Supplementary Table 1.

Growth and Oxygen Consumption Assays

Growing cultures of N gonorrhoeae were diluted to OD₆₀₀ 0.1 in fresh chemically defined medium (CDM) [8], supplemented as follows: 0.5% (w/v) D-glucose or 0.5% L-lactate as the sole carbon source, 0.5% (w/v) L-lactate and 100 μM FeSO₄.7H₂0 (Sigma) (+Fe environment), or 0.5% (w/v) L-lactate and 20 μM deferoxamine (Sigma) (−Fe environment). Bacteria were grown aerobically (37°C, 200 rpm), and OD₆₀₀ was measured hourly. Neisseria gonorrhoeae respiration was measured as previously described [4] using an Oxygraph oxygen electrode (Hansatech).

Quantitative Gene Expression Studies

Ribonucleic acid (RNA) was isolated from mid-exponential phase N gonorrhoeae (QIAGEN RNeasy Plus) and converted to complementary DNA (Superscript III; Invitrogen). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed using SYBR Green Master Mix (Applied Biosystems) in a ViiA7 real-time PCR machine (Life Technologies). Relative gene expression was calculated using the 2^−ΔCT method with 16S ribosomal RNA as a reference. The Fur-regulated genes, fbpA and bfrA, were used as controls. Primers used are described in Supplementary Table 1.

Neisseria gonorrhoeae Association, Invasion, and Survival Assays

The pex cells were procured from surgical cervical tissue in accordance with National Institutes of Health guidelines (article HHS45CFR46, exemption 4) and maintained as described previously [9]. Quantitative association, invasion, and survival assays were performed as previously described under microaerobic conditions [9].

Statistical Analysis

Differences in oxygen consumption, relative gene expression, and survival assays were analyzed using a 2-tailed unpaired t test and/or one-way analysis of variance (GraphPad Prism).

RESULTS

LldD and LutACB in Neisseria gonorrhoeae Are Both Involved in L-Lactate Utilization

To determine whether LutACB plays a role in Lut, growth assays were performed comparing 1291Δlut, 1291ΔlldD, and 1291ΔΔlut/lldD to the growth of the wild-type and the 1291ΔΔlut/lldD::lut-complemented mutant. The growth for the ΔlldD and Δlut mutants was similar to that of the wild-type strain on L-lactate. However, the ΔΔlut/lldD double mutant failed to grow on L-lactate, and complementation of this strain with lutACB restored growth to that of wild-type levels (Figure 1A). All strains grew equivalently on D-glucose (control) (Figure 1B), suggesting that NGO0904-0906 (lutACB) encodes a functional L-lactate dehydrogenase.

Figure 1.

Growth curve analysis and oxygen consumption when L-lactate or D-glucose was provided as the sole carbon, or electron donor, source. Assays were performed using Neisseria gonorrhoeae 1291 wild-type (WT), ΔlldD and Δlut deletion mutants, ΔΔlldD/lut double-deletion mutant, and ΔΔlldD/lut::lut-complemented mutant. Growth in chemically defined medium containing either L-lactate (A) or D-glucose (B) is shown. Carbon sources were added at a final concentration of 0.5% (w/v). Data shown are representative of at least 3 independent experiments. Oxygen consumption of N gonorrhoeae strains when either L-lactate (C) or D-glucose (D) was provided as the electron donor. Each assay was performed in triplicate on 3 separate occasions, and error bars represent the standard deviation of the 3 independent experiments. One-way analysis of variance was performed for WT versus each mutant and for the ΔΔlldD/lut double-deletion mutant versus the ΔΔlldD/lut::lut-complemented mutant. ****, P < .0001.

To confirm the function of LutACB, we measured the aerobic respiratory activities of the strains. A basal level of respiratory activity was observed for all the strains when D-glucose was the sole external electron donor (Figure 1D). Although both 1291ΔlldD and1291Δlut were able to respire using L-lactate, respiration was reduced when compared with that of 1291 wild-type (P < .0001) (Figure 1C). The ΔΔlut/lldD double mutant demonstrated an even greater reduction in respiration when compared with that of the single mutants (P < .0001) (Figure 1C). The severely compromised ability of 1291ΔΔlut/lldD to respire using L-lactate as an electron donor is indicative of the loss of both types of L-lactate dehydrogenases. Respiration could be partially restored upon complementation of 1291ΔΔlut/lldD::lut with lutACB (Figure 1C), thus confirming that LutACB encodes a functional L-lactate dehydrogenase in N gonorrhoeae.

LutACB Contributes to L-Lactate Dehydrogenase Activity Under Iron-Replete Conditions

Although LutACB and LldD function as L-lactate dehydrogenases, the former contains iron-sulfur clusters, whereas the latter is a flavoprotein that does not contain iron [4, 10]. These differences in prosthetic group composition, as well as the established knowledge that lutACB is part of the Fur regulon and is regulated by iron [7], prompted us to test the effect of iron on the growth of wild-type and mutants. We found that there was no difference in the growth of 1291 wild-type, 1291∆lldD, and 1291∆lutACB in CDM plus L-lactate, without (Supplementary Figure S1A) or with (Supplementary Figure S1B) the addition of iron. However, when iron was restricted, there was a reduction in the growth of the ΔlldD mutant when compared with the wild-type (Figure 2A and Supplementary Figure S1C for visual comparison). This indicated that iron-limitation was linked to a decrease in the L-lactate dehydrogenase capacity when lutACB was the only available L-lactate dehydrogenase.

Figure 2.

Growth curve analysis, oxygen consumption, and relative gene expression study with L-lactate under iron-replete and iron-limiting conditions. Growth curve analysis of Neisseria gonorrhoeae 1291 wild-type (WT), 1291ΔlldD, and 1291Δlut deletion mutants and, 1291ΔΔlldD/lut double-deletion mutant in chemically defined medium containing 0.5% (w/v) L-lactate and 20 μM deferoxamine, to create an iron-limiting environment (A). Data shown are representative of at least 3 independent experiments. Oxygen consumption by N gonorrhoeae 1291ΔlldD and 1291Δlut with L-lactate as the electron donor and in an iron-replete (+Fe) or iron-limiting (−Fe) condition (B). Each assay was performed in triplicate with error bars representing standard deviations. A one-way analysis of variance (ANOVA) was performed to determine the significance of oxygen consumed in L-lactate alone versus either the +Fe or −Fe condition. ****, P < .0001, ***, P < 0.001, and ns = not significant. Relative gene expression of lldD and lutA genes in either iron-replete (+Fe) or iron-limiting (−Fe) conditions (C). The error bars are indicative of the standard deviation of 3 independent experiments. The fold change in relative gene expression is shown for lldD or lutA in N gonorrhoeae when grown in either +Fe or −Fe conditions versus grown on L-lactate only. The relative gene expression was calculated using the 2^−ΔCT method, with 16S as the reference gene. A Student's t test was used to determine the significance of the relative change in lldD or lutA expression in either +Fe or −Fe condition versus the control (L-lactate only). ***, P < 0.001 and *, P < 0.05. Survival assay showing the effect of lactate dehydrogenase mutation on the ability of N gonorrhoeae strains 1291 WT, ΔlctP, ΔlldD, ΔlldD::lldD, Δlut, ΔΔlldD/lut, and ΔΔlldD/lut::lut to survive intracellularly within primary human cervical epithelial (pex) cells (D). A modified gentamycin-protection assay was performed under microaerobic conditions, and the mean (and variance) percentage (vs the inocula) of viable gonococci at 3 hours postgentamycin treatment is shown. A Kruskal-Wallis one-way ANOVA was used to determine the statistical significance of the ability of 1291WT versus each mutant to survive pex cell infection as well as the percentage survival of 1291ΔΔlldD/lut versus the 1291ΔΔlldD/lut::lut complement. ****, P < 0.0001 and ns = not significant.

To confirm these conclusions, we measured the effect of iron on L-lactate-dependent respiration in both the ΔlldD and Δlut mutants. Figure 2B demonstrates that iron restriction led to a significant decrease in L-lactate respiratory activity in 1291ΔlldD when compared with 1291Δlut (P < .0001). However, the opposite was true in the Δlut mutant in which a significant increase in L-lactate respiratory activity occurred when iron was restricted (P < .001) (Figure 2B). These observations are consistent with the suggestion that NGO0904-0906 (lutACB) is positively regulated by iron and Fur [7].

Iron Inversely Regulates lldD- and lutACB-Dependent Lactate Dehydrogenase Activities in Neisseria gonorrhoeae

Our data suggested that iron has opposite effects on the activity of LutACB and LldD, which partially substantiates a previous study describing that lutACB is regulated by iron [7]. We extended our analysis to determine the effect of iron on the expression of lldD and lutA using qRT-PCR. In the presence of iron, lldD gene expression was downregulated 1.5-fold (Figure 2C and Supplementary Figure S2). However, a significant 4-fold (P < .001) increase in lldD expression occurred under iron-limited conditions (Figure 2C and Supplementary Figure S2). This pattern of gene expression was similar to fbp, a gene that is known to be induced under conditions of iron limitation (Supplementary Figure S2) [7]. In contrast, lutA expression was upregulated in the presence of iron and downregulated under iron-limiting conditions (Figure 2C and Supplementary Figure S2), which is similar to bfr, a gene that is known to be positively regulated by iron (Supplementary Figure S2) [7]. Taken together, these data are the first to show that the expression of lldD is responsive to iron availability and confirm the role of iron in lut expression.

The lutACB Deletion Mutant Is Impaired in Its Ability to Survive Within Human Primary Cervical Epithelial Cells

We have shown that the intracellular survival of N gonorrhoeae under microaerobic conditions (3% O₂) is impaired when genes involved in lactate metabolism (lldD, ldhA, ldhD) are deleted [4]. Therefore, we performed the same infection studies to determine the role of LutACB in cervical infection (Figure 2D and Supplementary Figure S3). When compared with 1291 wild-type, there was no significant difference among any of the strains tested in their ability to associate with pex cells (Supplementary Figure S3). Furthermore, all strains exhibited similar gentamycin sensitivities except 1291ΔlctP, which was slightly more resistant (data not shown). A reduction in the invasive ability of 1291ΔlctP, 1291ΔlldD, and 1291ΔΔlut/lldD was observed, albeit these differences were modest and are unlikely to be biologically significant (Supplementary Figure S3). However, at 3 hours after gentamycin treatment, the ability of 1291Δlut, 1291ΔlldD, and 1291ΔΔlut/lldD to survive within pex cells was significantly reduced when compared with the 1291 wild-type strain. Moreover, this reduced survival was comparable to that observed for 1291ΔlctP, which lacks a functional lactate permease that is required for the uptake of L-lactate into N gonorrhoeae (P < .0001) (Figure 2D). The intracellular survival of the ΔlldD mutant was restored to wild-type levels when it was complemented with the lldD gene; however, complementation of 1291ΔΔlut/lldD with lutACB had a minimal, although significant, effect on gonococcal survival (P < .001) (Figure 2D). These data, taken together, indicate that whereas LutACB contributes to gonococcal survival during cervical infection, LldD appears to be essential to survival during in pex cell infection and/or under microaerobic conditions.

DISCUSSION

In this study, we demonstrated that LutACB encodes an L-lactate dehydrogenase in N gonorrhoeae in addition to the LldD described previously (Figure 1). Regulation of expression of these 2 types of L-lactate dehydrogenases by iron ensures that L-lactate metabolism is able to function independently of iron availability. This is of particular importance to host colonization. For example, humans possess iron transport and sequestration proteins such as transferrin, ferritin, hemoglobin, and Nramp-1 to transport, regulate, and withhold iron during infection [11]. In addition, iron availability within the female reproductive tract varies with the menstrual cycle [12].

The ability to grow intracellularly is shown to influence N gonorrhoeae survival, allowing protection from host defences and from competition with other bacteria [13]. Consistent with our previous study [4], we have now shown that LldD, as well as Lut and LctP, are critical for the intracellular survival and growth of N gonorrhoeae in pex cells. These results strongly suggest that L-lactate is a primary carbon source for intracellular N gonorrhoeae. This idea is consistent with our previous works that demonstrate a critical role for (host) Akt kinase in gonococcal infection of pex cells [13]. In this regard, one function of Akt kinase is to promote aerobic glycolysis, which results in lactate accumulation and cellular adaptation to a hypoxic environment [14].

CONCLUSIONS

We showed that iron availability is a factor affecting L-lactate dehydrogenase expression in N gonorrhoeae in contrast to Escherichia coli where lactate metabolic capability is controlled by LctR (lactate-dependent transcriptional regulator) [10]. It seems likely that N gonorrhoeae is almost always in contact with L-lactate; the variable is the availability of iron. Previous studies have shown that the lut operon is positively regulated by iron and Fur [7], and this transcriptional regulator also directly controls expression of the NADH dehydrogenase (Nuo) in N gonorrhoeae and Neisseria meningitidis and indirectly controls succinate dehydrogenase (Sdh) expression via the small RNA, NrrF [7, 15]. Given the pivotal role of Fur, we identified a putative Fur binding site in the lldD promoter region (Supplementary Figure S4), suggesting that Fur mediates iron-dependent repression of lldD. In summary, we have shown that lutACB encodes a functional lactate dehydrogenase that, together with LldD, may promote the intracellular survival of N gonorrhoeae during cervical infection.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Financial support. This research was funded by Australian Research Council Grant DP 140102130 (to A. G. M.) and by the National Institutes of Health Grant R01AI134848 (to J. L. E.) C.-l. Y. O. is supported by a Garnett Passe and Rodney Williams Memorial Foundation Research Fellowship. Cervical tissue samples were obtained from the Cooperative Human Tissue Network, which is funded by the National Cancer Institute.

Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest