Transcriptional responses of Neisseria gonorrhoeae to glucose and lactate: implications for resistance to oxidative damage and biofilm formation

Abstract

Understanding how bacteria adapt to different environmental conditions is crucial for advancing knowledge regarding pathogenic mechanisms that operate during infection as well as efforts to develop new therapeutic strategies to cure or prevent infections. Here, we investigated the transcriptional response of Neisseria gonorrhoeae, the causative agent of gonorrhea, to L-lactate and glucose, two important carbon sources found in the host environment. Our study revealed extensive transcriptional changes that gonococci make in response to L-lactate, with 37% of the gonococcal transcriptome being regulated, compared to only 9% by glucose. We found that L-lactate induces a transcriptional program that would negatively impact iron transport, potentially limiting the availability of labile iron, which would be important in the face of the multiple hydrogen peroxide attacks encountered by gonococci during its lifecycle. Furthermore, we found that L-lactate-mediated transcriptional response promoted aerobic respiration and dispersal of biofilms, contrasting with an anaerobic condition previously reported to favor biofilm formation. Our findings suggest an intricate interplay between carbon metabolism, iron homeostasis, biofilm formation, and stress response in N. gonorrhoeae, providing insights into its pathogenesis and identifying potential therapeutic targets.IMPORTANCEGonorrhea is a prevalent sexually transmitted infection caused by the human pathogen Neisseria gonorrhoeae, with ca. 82 million cases reported worldwide annually. The rise of antibiotic resistance in N. gonorrhoeae poses a significant public health threat, highlighting the urgent need for alternative treatment strategies. By elucidating how N. gonorrhoeae responds to host-derived carbon sources such as L-lactate and glucose, this study offers insights into the metabolic adaptations crucial for bacterial survival and virulence during infection. Understanding these adaptations provides a foundation for developing novel therapeutic approaches targeting bacterial metabolism, iron homeostasis, and virulence gene expression. Moreover, the findings reported herein regarding biofilm formation and L-lactate transport and metabolism contribute to our understanding of N. gonorrhoeae pathogenesis, offering potential avenues for preventing and treating gonorrhea infections.

Keywords: H2O2 resistance; Neisseria gonorrhoeae; iron transport; lactate utilization.

Fig 1

Genome-wide transcriptional landscape changes of N. gonorrhoeae to L-lactate and glucose in the culture medium. (A) Graphic representation of gonococcal genes activated (green) and repressed (red) by glucose and L-lactate determined by RNA-Seq. N. gonorrhoeae cells were grown in GC broth at 1 or 10 mM of each sugar and collected at the mid-logarithmic phase for RNA-Seq. The numbering indicates the chromosome coordinates in kb pairs. The black plot is the deviation from the average GC content of the genome. GGI: gonococcal genetic island. (B) Venn diagram of glucose and L-lactate co-regulated genes (at fold-change ≥2). (C) The number of glucose and L-lactate up- and down-regulated genes (at fold-change ≥2).

Fig 2

Validation of the RNA-Seq study using qRT-PCR in a subset of 10 and 14 genes for the glucose and L-lactate regulon, respectively. A subset of statistically significant up- or down-regulated genes (except for recA, which is housekeeping gene not regulated) for the observed glucose and L-lactate regulons were selected for qRT-PCR validation. N. gonorrhoeae FA19 was grown in GC-broth containing 1 or 10 mM of glucose with a constant concentration of 3 mM L-lactate, or 1 or 10 mM of L-lactate with a constant concentration of 1.5 mM glucose. Cells were collected at the mid-logarithmic phase for RNA extraction. Each gene point is scattered based on the fold-change (FC) regulation between 1 and 10 mM of each sugar. The nonparametric Spearman correlation coefficients (R) and P values (two-tailed) are indicated for the glucose (left) and L-lactate (right) transcriptomic analyses.

Fig 3

Representation of up- and down-regulated genes within the glucose (A) and L-lactate (B) RNA-seq-determined regulons grouped by functional categories. Shown on the y-axis is the number of genes within a functional category (x-axis) that were regulated ≥ two-fold.

Fig 4

Correlation analysis of the L-lactate and glucose RNA-Seq-determined regulons with the regulon of different conditions faced by the gonococci during infection. Venn diagrams were drawn depicting the relationship of the L-lactate and glucose regulons with previously determined transcriptomes for sub-lethal hydrogen peroxide exposure [using the bioinformatic analysis compiled by (28) from original data reported by (14)] (A), anaerobic conditions (13) (B) and iron-replete conditions [using bioinformatic analysis compiled by (28) from original data reported by (11)] (C). Co-regulated genes between the L-lactate regulon and each of the three previously stated experimental conditions in a, b, and c were used to construct scatter plot graphs using the fold-change regulation as x,y variables and for correlation analysis (D,E,F). Correlation analysis between the glucose regulon and the regulon of the three experimental conditions are presented in (G,H,I). The nonparametric Spearman coefficient (R) and P values are indicated for each correlation analysis.

Fig 5

The subset of co-regulated genes between the L-lactate, iron-replete conditions, and sub-lethal hydrogen peroxide exposure regulons contains mainly iron transport and iron-sulfur cluster repair genes. FC: fold-change. Please see the legend in Fig. 4 for the source of data for the regulons described in the figure.

Fig 6

Effect of L-lactate on gonococcal biofilms. Neisseria gonorrhoeae strains FA19 and F62 were grown in GC broth containing either glucose (22 mM) or L-lactate (22 mM) in 96-well plates. Biofilm formation was quantified using the crystal violet staining method. The data are represented as the median (bar) plus/minus the 75% and 25% percentiles, respectively (error bars) of 4 biological replicates for FA19 and 3 for F62. For both strains, the difference in biofilm level in glucose vs L-lactate-grown gonococci was statistically significant using a nonparametric Mann-Whitney U-test (one-tailed P = 0.0143 for FA19 and P = 0.05 for F62).

Fig 7

Gonococcal cells lacking lctP have a significant growth defect in GC-broth supplemented with pyruvate in replacement of glucose. Wild-type gonococcal strain FA19 and isogenic strains gdhR insertional mutant (FA19 gdhR::kan), complemented strain overexpressing gdhR in trans from the IPTG-inducible lac promoter (JC02), lctP insertional mutant (JC03), and double mutant gdhR-lctP (JC04) were grown in GC-broth supplemented with pyruvate in replacement of glucose in the Kellogg supplement (A) and with standard glucose-containing Kellogg supplement (B). Bacteria were grown with orbital shaking at 37°C for 6 h and the optical density (OD) at 600 nm was measured every hour. Data are represented as the median plus/minus the 75% and 25% percentiles (error bars), respectively, of three biological replicates. *1 statistically significant at 6 h from the WT using a nonparametric Mann-Whitney U-test (one-tailed P = 0.05). *2 statistically significant at 6 h from the rest of the strains using a U-test (one-tailed P = 0.05). ns non-significant statistical differences at 6 h from wild type using a one-tailed U-test.

Fig 8

Proposed model for the mechanisms by which L-lactate transport and metabolism protect gonococcal cells from hydrogen peroxide-induced oxidative damage and promote survival within infection sites. Bacterial cells tightly control the level of free cytoplasmatic iron using the repressive action of the Holo-Fur dimer on iron uptake genes (11). This is because unbound ferrous iron catalyzes the conversion of the non-toxic H₂O₂ into damaging hydroxyl radicals by the Fenton reaction. We call the Fur protection Layer-1. However, in the presence of increasing concentration of H₂O₂, the ferrous iron clusters of Fur are oxidized to ferric iron by the Fenton reaction, resulting in the inability of Fur to repress the transcription of iron-uptake genes (14). We have shown that increasing concentrations of H₂O₂ in the presence of ferrous iron can attack and lead to a decrease in the formation of repressive GdhR-lctP nucleoprotein complexes, which increases lctP expression (18). Increased expression of LctP at the inner membrane (IM) can lead to an increased influx of L-lactate and pyruvate. Pyruvate is a direct H₂O₂ scavenger by undergoing a decarboxylation reaction (38) and protects bacterial cells from H₂O₂ killing (59). Increased L-lactate transport results in the repression of iron-uptake genes, which protects gonococcal cells from H₂O₂-induced oxidative damage. In addition, it increases the expression of genes encoding the tricarboxylic acid cycle, respiratory enzymes, and ribosomal proteins, which leads to an increase in oxygen consumption and acceleration of the metabolic activity allowing gonococci to effectively compete with neutrophils for oxygen and to resist the respiratory burst (26, 40, 41, 49). L-lactate induced transcription of pilE, encoding the main type IV pilus subunit, and piliation is required for resistance to H₂O₂- and neutrophil-mediated killing (60). We propose that the L-lactate regulon-induced effects constitute a layer-2 protection system from oxidative damage complimentary to the Fur regulation system and that it should take place at high levels of hydrogen peroxide exposure such as within the phagolysosome of polymorphonuclear cells. L-lactate was shown to effectively disrupt gonococcal biofilm formation, which could be important for the initial attachment and colonization of the genital sites (i.e., within the vaginal tract colonized by lactic acid-producing lactobacilli). The illustration was designed with BioRender.com.