SummaryThe initial attachment of Neisseria meningitidis to the target cell surface appears to be largely pilus dependent in capsulated bacteria. Intimate adhesion subsequently occurs to permit colonization. We recently reported that insertional inactivation of the crgA gene, which encodes a transcriptional regulator belonging to the LysR family, decreased meningococcal adhesion to epithelial cells and abolished intimate adhesion. In this report, we analyse expression of the pilE and sia genes, which are involved in the biosynthesis of pili and capsule respectively, during bacteria-host cell interactions. Western blotting, transcriptional fusion and reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression of these genes was downregulated during intimate adhesion. DNA-binding assays, footprinting and RT-PCR analysis indicated that this downregulation was directly mediated by the CrgA protein. The pilE and sia promoters were found to have a CrgA binding motif in common. These results strongly suggest that N. meningitidis displays an adaptive response upon cell contact. CrgA may play a central regulatory role in meningococcal adhesion, particularly in switching from initial to intimate adhesion by downregulating the bacterial surface structures that hinder this adhesion.

PilC1, a pilus‐associated protein in Neisseria meningitidis, is a key element in initial meningococcal adhesion to target cells. A promoter element (CREN, contact regulatory element of Neisseria) is responsible for the transient induction of this gene upon cell contact. crgA (contact‐regulated gene A) encodes a transcriptional regulator whose expression is also induced upon cell contact from a promoter region similar to the CREN of pilC1. CrgA shows significant sequence homologies to LysR‐type transcriptional regulators. Its inactivation in meningococci provokes a dramatic reduction in bacterial adhesion to epithelial cells. Moreover, this mutant is unable to undergo intimate adhesion to epithelial cells or to provoke effacing of microvilli on infected cells. Purified CrgA is able to bind to pilC1 and crgA promoters, and CrgA seems to repress the expression of pilC1 and crgA. Our results support a dynamic model of bacteria–cell interaction involving a network of regulators acting in cascade. CrgA could be an intermediate regulator in such a network.

SummaryPilus-mediated adherence makes an essential contribution to the pathogenesis of Neisseria meningitidis by allowing the initial localized adherence. Pili are assembled from a protein subunit called pilin. Two proteins, PilC1 and PilC2, are also key elements in the formation of pili as the production of at least one PilC protein is required for pilus assembly. In addition, PilC1 but not PilC2 modulates adhesiveness, most probably by being the adhesin. Recently, both genes have been demonstrated to be controlled by different promoters, pilC2 is expressed from a single transcription starting point (TSP), whereas pilC1 has three TSPs. One of these, PC1.1, corresponds to the unique TSP of pilC2, and two others, PC1.2 and PC1.3, are located in a region upstream of pilC1 but not pilC2. This suggests that both genes may be under the control of separate regulatory pathways. In this work, by engineering pilC1-lacZ and pilC2-lacZ transcriptional fusions, we provide evidence that expression of pilC1, but not that of pilC2, is transiently induced by bacterial cell contact. This induction required viable cells, did not need the presence of pili and relied on the expression of pilC1 from PC1.3. Destruction of this TSP by site-directed mutagenesis did not significantly diminish the piliation level or the basal expression of PilC1, but led to the loss of cell contact-dependent upregulation of pilC1 and to a dramatic decrease in bacterial adhesiveness. Taken together, these data demonstrate that cell contact-dependent upregulation of the transcription of pilC1 at PC1.3 is essential for meningococcal pilusmediated adhesion.

Bacteria have developed several mechanisms which allow the preferred utilization of the most efficiently metabolizable carbohydrates when these organisms are exposed to a mixture of carbon sources. Interestingly, the same or similar mechanisms are used by some pathogens to control various steps of their infection process. The efficient metabolism of a carbon source might serve as signal for proper fitness. Alternatively, the presence of a specific carbon source might indicate to bacterial cells that they thrive in infection-related organs, tissues or cells and that specific virulence genes should be turned on or switched off. Frequently, virulence gene regulators are affected by changes in carbon source availability. For example, expression of the gene encoding the Streptococcus pyogenes virulence regulator Mga is controlled by the classical carbon catabolite repression (CCR) mechanism operative in Firmicutes. The activity of PrfA, the major virulence regulator in Listeria monocytogenes, seems to be controlled by the phosphorylation state of phosphotransferase system(PTS) components. In Vibrio cholerae synthesis of HapR, which regulates the expression of genes required for motility, is controlled via the Crp/cAMP CCR mechanism, whereas synthesis of Salmonella enterica HilE, which represses genes in a pathogenicity island, is regulated by the carbohydrate-responsive, PTS-controlled Mlc.

Phosphorylation and dephosphorylation at Ser-46 in HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is controlled by the bifunctional HPr kinase/phosphorylase (HprK/P). In Gram-positive bacteria, P-Ser-HPr controls (1) sugar uptake via the PTS; (2) catabolite control protein A (CcpA)-mediated carbon catabolite repression, and (3) inducer exclusion. Genome sequencing revealed that HprK/P is absent from Gram-negative enteric bacteria, but present in many other proteobacteria. These organisms also possess (1) HPr, the substrate for HprK/P; (2) enzyme I, which phosphorylates HPr at His-15, and (3) one or several enzymes IIA, which receive the phosphoryl group from P∼His-HPr. The genes encoding the PTS proteins are often organized in an operon with hprK. However, most of these organisms miss CcpA and a functional PTS, as enzymes IIB and membrane-integrated enzymes IIC seem to be absent. HprK/P and the rudimentary PTS phosphorylation cascade in Gram-negative bacteria must therefore carry out functions different from those in Gram-positive organisms. The gene organization in many HprK/P-containing Gram-negative bacteria as well as some preliminary experiments suggest that HprK/P might control transcription regulators implicated in cell adhesion and virulence. In α-proteobacteria, hprK is located downstream of genes encoding a two-component system of the EnvZ/OmpR family. In several other proteobacteria, hprK is organized in an operon together with genes from the rpoN region of Escherichia coli (rpoN encodes a σ54). We propose that HprK/P might control the phosphorylation state of HPr and EIIAs, which in turn could control the transcription regulators.