Coordinated Transcriptional Increases in Cell Wall Synthesis Genes in Neisseria gonorrhoeae Lacking the Lytic Transglycosylase, ltgA

Abstract

Lytic transglycosylase A in Neisseria gonorrhoeae cleaves the β-1,4-glycosidic bond between peptidoglycan (PG) monomers to liberate 1,6-anhydro-PG fragments that are either recycled or released as cytotoxic fragments. To gain further insight into the effect of LtgA on cellular processes in Neisseria gonorrhoeae, we performed a proteomic analysis comparing wild-type and an isogenic ltgA null mutant strain. Proteins were separated by two-dimensional gel electrophoresis and identified by MALDI-TOF mass spectrometry, which revealed several proteins that were increased in their level of expression upon loss of LtgA. The most notable changes corresponded to enzymes related to aminosugar and pyrimidine metabolism. Quantitative real-time RT-PCR of mRNA from a ltgA null strain confirmed increased transcription of genes encoding enzymes involved in UDP-N-acetylglucosamine (UDP-GlcNAc) synthesis, a major precursor in PG and lipooligosaccharide (LOS) synthesis, during normal growth conditions and following exposure to penicillin. We also found that the ltgA mutant strains were more susceptible to β-lactam antibiotics, vancomycin, and the human-cathelicidin antibacterial peptide, LL-37, than their corresponding wild-type parental strains. Our results suggest that increased expression of enzymes responsible for production UDP-GlcNAc is an adaptive response due to inactivation of ltgA and/or exposure to penicillin.

Fig. 1

Two-dimensional gel of proteins isolated from exponential and stationary phase gonococcal cultures. Panels are sections from the total protein profile image generated. Circled spots indicate proteins that were altered in expression between FAl9 wild-type (panel a) and LW9279 (panel b) during exponential and stationary phase. The circled spots were identified by mass spectrometry (Table 2 and S2)

Fig. 2

Biosynthetic pathway for production of UDP-GlcNAc

Fig. 3

Expression of orf NG0373, glmS, and glmU in strains, a LW9279 (FA19∆ltgA) and b KC100 (MS11∆ltgA), during stationary phase. Transcript levels were measured by qRT-PCR and indicate a change in expression levels in mutant relative to its respective parent strain. P values and SEM were determined from results of three or more independent experiments

Fig. 4

a Expression of a putative ABC transporter (NG0373), glmS, glmU, and uprT during stationary phase in KC100 (MS11∆ltgA) relative to MS11 wild-type. Orf NG0373 and glmU mRNA-transcript levels were restored back to wild-type level whereas glmS and uprT mRNA-transcript levels were restored back to a near wild-type level in strain KH599 (ltgA complement strain). A P value of < 0.0001 was determined and the SEM calculated from results of one representative experiment. b uprT expression during stationary phase in FA19 and MS11 wild-type strains and isogenic mutant ltgA strains (LW9279 and KC100, respectively). Transcript levels for uprT were measured by qRT-PCR and indicate the relative change in expression of uprT in mutant ltgA strains relative to wild-type. A P value of < 0.0001 was determined and SEM calculated from two independent experiments

Fig. 5

Expression of ponA in a mutant ltgA strain. There was a 3.5-fold (P = 0.0194) increase in ß-galactosidase activity in LW9279 (FA19 ∆ltgA) relative to FA19 wildtype. The results are shown as the average values with a calculated stand error of the mean (SEM) determined from four independent assays

Fig. 6

Relative expression levels of ponA in mutant ltgA. qPCR results show that ponA mRNA-transcript levels were increased in strain LW9279 relative to FA19 wildtype. A P value of 0.04 indicates a significant difference in expression levels. The results are shown as the average values with a calculated stand error of the mean (SEM) determined from four independent assays

Fig. 7

The effect of N-acetyl-D-glucosamine (UDP-GlcNAc) on orf NG0373, glmS, glmU, and uprT mRNA-transcript levels. The addition of 50 mM UDP-GlcNAc to KC100 (+ DP-GlcNAc) restored gene expression to a near wild-type levels. P values (located above bar) were determined-based replicates of three for each gene of at least two or more independent experiments

Fig. 8

Relative expression levels of (a). glmS and (b). uprT for penicillin (+ PEN) treated strains relative to MS11-untreated (−PEN). Transcript levels were measured by qRT-PCR and indicate the relative change in gene expression between strains. A P value of 0.014 and 0.001 was calculated for glmS and uprT, respectively. Values and the SEM were calculated from the results of three independent experiments