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Host cell-derived lactate functions as an effector molecule in Neisseria meningitidis microcolony dispersal

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ABSTRACT

The development of meningococcal disease, caused by the human pathogen Neisseria meningitidis, is preceded by the colonization of the epithelial layer in the nasopharynx. After initial adhesion to host cells meningococci form aggregates, through pilus-pilus interactions, termed microcolonies from which the bacteria later detach. Dispersal from microcolonies enables access to new colonization sites and facilitates the crossing of the cell barrier; however, this process is poorly understood. In this study, we used live-cell imaging to investigate the process of N. meningitidis microcolony dispersal. We show that direct contact with host cells is not required for microcolony dispersal, instead accumulation of a host-derived effector molecule induces microcolony dispersal. By using a host-cell free approach, we demonstrated that lactate, secreted from host cells, initiate rapid dispersal of microcolonies. Interestingly, metabolic utilization of lactate by the bacteria was not required for induction of dispersal, suggesting that lactate plays a role as a signaling molecule. Furthermore, Neisseria gonorrhoeae microcolony dispersal could also be induced by lactate. These findings reveal a role of host-secreted lactate in microcolony dispersal and virulence of pathogenic Neisseria.

No MeSH data available.


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Expression of genes and proteins associated with N. meningitidis colonization upon addition of CM.The gene expression and protein level was examined upon addition of CM to preformed FAM20 microcolonies, with DMEM used as a control. (A) For quantitative real-time PCR, samples were taken 10 min after addition of CM. Target mRNA levels were normalized to the housekeeping gene coding for the 30S ribosomal protein RpsJ. (B) For Western blot analysis, samples were taken 10 min after addition of CM. After detection of the protein of interest, the membrane was stripped and the expression of EF-Tu was examined and used as a loading control. Gene and protein expression levels in the controls were set to a value of 1. Data represent the mean ± SD of three independent experiments. *p < 0.05. ns, non-significant.
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ppat.1006251.g003: Expression of genes and proteins associated with N. meningitidis colonization upon addition of CM.The gene expression and protein level was examined upon addition of CM to preformed FAM20 microcolonies, with DMEM used as a control. (A) For quantitative real-time PCR, samples were taken 10 min after addition of CM. Target mRNA levels were normalized to the housekeeping gene coding for the 30S ribosomal protein RpsJ. (B) For Western blot analysis, samples were taken 10 min after addition of CM. After detection of the protein of interest, the membrane was stripped and the expression of EF-Tu was examined and used as a loading control. Gene and protein expression levels in the controls were set to a value of 1. Data represent the mean ± SD of three independent experiments. *p < 0.05. ns, non-significant.

Mentions: In response to CM, we detected significant changes in expression of the pilus-associated genes pilE, pilT, pilC1, pilC2, pilX, pilV, pilW, pptB [4, 7, 9, 14, 25, 26] and the transcriptional regulator crgA [27, 28], (Fig 3A). However, no significant changes were observed in expression of the transcriptional regulators misR or pnp [8, 29]. Additionally, no significant changes were observed in the genes pglC, pglI, pglB2, pglH and pglL, encoding for pilus posttranslational modification enzymes [30], (Fig 3A). Moreover, we analyzed the protein level of PilE, PilT, PilC, PilX and PilW at 10 min after addition of CM to microcolonies. We did not observe any significant changes on the protein level upon addition of CM (Fig 3B). The results indicate that expression of the proteins PilE, PilT, PilC, PilX and PilW, previously associated with meningococcal colonization, was not changed upon induction of microcolony dispersal. To summarize, these results indicate that N. meningitidis in microcolonies disperses in response to one or more compounds derived from host cells. The activity of the CM increased with incubation time, suggesting that the active molecule(s) accumulate over time.


Host cell-derived lactate functions as an effector molecule in Neisseria meningitidis microcolony dispersal
Expression of genes and proteins associated with N. meningitidis colonization upon addition of CM.The gene expression and protein level was examined upon addition of CM to preformed FAM20 microcolonies, with DMEM used as a control. (A) For quantitative real-time PCR, samples were taken 10 min after addition of CM. Target mRNA levels were normalized to the housekeeping gene coding for the 30S ribosomal protein RpsJ. (B) For Western blot analysis, samples were taken 10 min after addition of CM. After detection of the protein of interest, the membrane was stripped and the expression of EF-Tu was examined and used as a loading control. Gene and protein expression levels in the controls were set to a value of 1. Data represent the mean ± SD of three independent experiments. *p < 0.05. ns, non-significant.
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Related In: Results  -  Collection

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ppat.1006251.g003: Expression of genes and proteins associated with N. meningitidis colonization upon addition of CM.The gene expression and protein level was examined upon addition of CM to preformed FAM20 microcolonies, with DMEM used as a control. (A) For quantitative real-time PCR, samples were taken 10 min after addition of CM. Target mRNA levels were normalized to the housekeeping gene coding for the 30S ribosomal protein RpsJ. (B) For Western blot analysis, samples were taken 10 min after addition of CM. After detection of the protein of interest, the membrane was stripped and the expression of EF-Tu was examined and used as a loading control. Gene and protein expression levels in the controls were set to a value of 1. Data represent the mean ± SD of three independent experiments. *p < 0.05. ns, non-significant.
Mentions: In response to CM, we detected significant changes in expression of the pilus-associated genes pilE, pilT, pilC1, pilC2, pilX, pilV, pilW, pptB [4, 7, 9, 14, 25, 26] and the transcriptional regulator crgA [27, 28], (Fig 3A). However, no significant changes were observed in expression of the transcriptional regulators misR or pnp [8, 29]. Additionally, no significant changes were observed in the genes pglC, pglI, pglB2, pglH and pglL, encoding for pilus posttranslational modification enzymes [30], (Fig 3A). Moreover, we analyzed the protein level of PilE, PilT, PilC, PilX and PilW at 10 min after addition of CM to microcolonies. We did not observe any significant changes on the protein level upon addition of CM (Fig 3B). The results indicate that expression of the proteins PilE, PilT, PilC, PilX and PilW, previously associated with meningococcal colonization, was not changed upon induction of microcolony dispersal. To summarize, these results indicate that N. meningitidis in microcolonies disperses in response to one or more compounds derived from host cells. The activity of the CM increased with incubation time, suggesting that the active molecule(s) accumulate over time.

View Article: PubMed Central - PubMed

ABSTRACT

The development of meningococcal disease, caused by the human pathogen Neisseria meningitidis, is preceded by the colonization of the epithelial layer in the nasopharynx. After initial adhesion to host cells meningococci form aggregates, through pilus-pilus interactions, termed microcolonies from which the bacteria later detach. Dispersal from microcolonies enables access to new colonization sites and facilitates the crossing of the cell barrier; however, this process is poorly understood. In this study, we used live-cell imaging to investigate the process of N. meningitidis microcolony dispersal. We show that direct contact with host cells is not required for microcolony dispersal, instead accumulation of a host-derived effector molecule induces microcolony dispersal. By using a host-cell free approach, we demonstrated that lactate, secreted from host cells, initiate rapid dispersal of microcolonies. Interestingly, metabolic utilization of lactate by the bacteria was not required for induction of dispersal, suggesting that lactate plays a role as a signaling molecule. Furthermore, Neisseria gonorrhoeae microcolony dispersal could also be induced by lactate. These findings reveal a role of host-secreted lactate in microcolony dispersal and virulence of pathogenic Neisseria.

No MeSH data available.


Related in: MedlinePlus