The ciaRH operon in Streptococcus mutans contains 3 contiguous genes, ciaXRH. Unlike the CiaRH system in other streptococci, only the ciaH-null mutant displays defective phenotypes, while the ciaR-null mutant behaves like the wild type. The objective of this study was to determine the mechanism of this unusual property. We demonstrate that the ciaH mutation caused a >20-fold increase in ciaR transcript synthesis. A ciaRH double deletion reversed the ciaH phenotype, suggesting that overexpressed ciaR might be responsible for the observed ciaH phenotypes. When ciaR was forced to be overexpressed by a transcriptional fusion to the ldh promoter in the wild-type background, the same ciaH phenotypes were restored, confirming the involvement of overexpressed ciaR in the ciaH phenotypes. The ciaH mutation and ciaR overexpression also caused transcriptional alterations in 100 genes, with 15 genes upregulated >5-fold. Bioinformatics analysis identified a putative CiaR regulon consisting of 8 genes/operons, including the ciaXRH operon itself, all of which were upregulated. In vitro footprinting on 4 of the 8 promoters revealed a protected region of 26 to 28 bp encompassing two direct repeats, NTTAAG-n5-WTTAAG, 10 bp upstream of the −10 region, indicating direct binding of the CiaR protein to these promoters. Taken together, we conclude that overexpressed CiaR, as a result of either ciaH deletion or forced expression from a constitutive promoter, is a mediator in the CiaH-regulated phenotypes.
The ciaR-ciaH system is one of 13 two-component signal-transducing systems of the human pathogen Streptococcus pneumoniae. Mutations in the histidine protein kinase CiaH confer increased resistance to beta-lactam antibiotics and interfere with the development of genetic competence. In order to identify the genes controlled by the cia system, the cia regulon, DNA fragments targeted by the response regulator CiaR were isolated from restricted chromosomal DNA using the solid-phase DNA binding assay and analyzed by hybridization to an oligonucleotide microarray representing the S. pneumoniae genome. A set of 18 chromosomal regions containing 26 CiaR target sites were detected and proposed to represent the minimal cia regulon. The putative CiaR target loci included genes important for the synthesis and modification of cell wall polymers, peptide pheromone and bacteriocin production, and the htrA-spo0J region. In addition, the transcription profile of cia loss-of-function mutants and those with an apparent activated cia system representing the off and on states of the regulatory system were analyzed. The transcript analysis confirmed the cia-dependent expression of seven putative target loci and revealed three additional cia-regulated loci. Five putative target regions were silent under all conditions, and for the remaining three regions, no cia-dependent expression could be detected. Furthermore, the competence regulon, including the comCDE operon required for induction of competence, was completely repressed by the cia system.
Activation of the CiaRH two-component signaling system prevents the development of competence for genetic transformation in Streptococcus pneumoniae through a previously unknown mechanism. Earlier studies have shown that CiaRH controls the expression of htrA, which we show encodes a surface-expressed serine protease. We found that mutagenesis of the putative catalytic serine of HtrA, while not impacting the competence of a ciaRH+ strain, restored a normal competence profile to a strain having a mutation that constitutively activates the CiaH histidine kinase. This result implies that activity of HtrA is necessary for the CiaRH system to inhibit competence. Consistent with this finding, recombinant HtrA (rHtrA) decreased the competence of pneumococcal cultures. The rHtrA-mediated decline in transformation efficiency could not be corrected with excess competence-stimulating peptide (CSP), suggesting that HtrA does not act through degradation of this signaling molecule. The inhibitory effects of rHtrA and activated CiaH, however, were largely overcome in a strain having constitutive activation of the competence pathway through a mutation in the cytoplasmic domain of the ComD histidine kinase. Although these results suggested that HtrA might act through degradation of the extracellular portion of the ComD receptor, Western immunoblots for ComD did not reveal changes in protein levels attributable to HtrA. We therefore postulate that HtrA may act on an unknown protein target that potentiates the activation of the ComDE system by CSP. These findings suggest a novel regulatory role for pneumococcal HtrA in modulating the activity of a two-component signaling system that controls the development of genetic competence.
The two-component signal-transducing system CiaRH of Streptococcus pneumoniae plays an important role during the development of beta-lactam resistance in laboratory mutants. We show here that a functional CiaRH system is required for survival under many different lysis-inducing conditions. Mutants with an activated CiaRH system were highly resistant to lysis induced by a wide variety of early and late cell wall inhibitors, such as cycloserine, bacitracin, and vancomycin, and were also less susceptible to these drugs. In contrast, loss-of-function CiaRH mutants were hypersusceptible to these drugs and were apparently unable to maintain a stationary growth phase in normal growth medium and under choline deprivation as well. Moreover, disruption of CiaR in penicillin-resistant mutants with an altered pbp2x gene encoding low-affinity PBP2x resulted in severe growth defects and rapid lysis. This phenotype was observed with pbp2x genes containing point mutations selected in the laboratory and with highly altered mosaic pbp2x genes from penicillin-resistant clinical isolates as well. This documents for the first time that PBP2x mutations required for development of beta-lactam resistance are functionally not neutral and are tolerated only in the presence of the CiaRH system. This might explain why cia mutations have not been observed in penicillin-resistant clinical isolates. The results document that the CiaRH system is required for maintenance of the stationary growth phase and for prevention of autolysis triggered under many different conditions, suggesting a major role for this system in ensuring cell wall integrity.
Post-transcriptional regulation by small RNAs (sRNAs) in bacteria is now recognized as a wide-spread regulatory mechanism modulating a variety of physiological responses including virulence. In Streptococcus pneumoniae, an important human pathogen, the first sRNAs to be described were found in the regulon of the CiaRH two-component regulatory system. Five of these sRNAs were detected and designated csRNAs for cia-dependent small RNAs. CiaRH pleiotropically affects β-lactam resistance, autolysis, virulence, and competence development by yet to be defined molecular mechanisms. Since CiaRH is highly conserved among streptococci, it is of interest to determine if csRNAs are also included in the CiaRH regulon in this group of organisms consisting of commensal as well as pathogenic species. Knowledge on the participation of csRNAs in CiaRH-dependent regulatory events will be the key to define the physiological role of this important control system.
Genes for csRNAs were predicted in streptococcal genomes and data base entries other than S. pneumoniae by searching for CiaR-activated promoters located in intergenic regions that are followed by a transcriptional terminator. 61 different candidate genes were obtained specifying csRNAs ranging in size from 51 to 202 nt. Comparing these genes among each other revealed 40 different csRNA types. All streptococcal genomes harbored csRNA genes, their numbers varying between two and six. To validate these predictions, S. mitis, S. oralis, and S. sanguinis were subjected to csRNA-specific northern blot analysis. In addition, a csRNA gene from S. thermophilus plasmid pST0 introduced into S. pneumoniae was also tested. Each of the csRNAs was detected on these blots and showed the anticipated sizes. Thus, the method applied here is able to predict csRNAs with high precision.
The results of this study strongly suggest that genes for small non-coding RNAs, csRNAs, are part of the regulon of the two-component regulatory system CiaRH in all streptococci.
Oxygen controls competence development in Streptococcus pneumoniae. Oxygen signaling involves the two-component signal transduction systems CiaRH and ComDE and the competence-stimulating peptide encoded by comC and processed by ComAB. We found that NADH oxidase (Nox) was required for optimal competence. Transcriptional analysis and genetic dissection showed that Nox was involved in post-transcriptional activation of the response regulator ComE and in the transcriptional control of ciaRH and comCDE. Thus, in S. pneumoniae, Nox, with O2 as its secondary substrate, is part of the O2-signaling pathway.
To assess potential function of each two-component signal transduction system in the expression of Streptococcus mutans virulence properties.
Methods and Results
For each two-component system (TCS), the histidine kinase-encoding gene was inactivated by a polymerase chain reaction (PCR)-based deletion strategy and the effects of gene disruption on the cell's ability to form biofilms, become competent, and tolerate acid, osmotic, and oxidative stress conditions were tested. Our results demonstrated that none of the mutations were lethal for S. mutans. The TCS-2 (CiaRH) is involved in biofilm formation and tolerance to environmental stresses, the TCS-3 (ScnRK-like) participates in the survival of cells at acidic pH, and the TCS-9 affects the acid tolerance response and the process of streptococcal competence development.
Our results confirmed the physiological role of the TCS in S. mutans cellular function, in particular the SncRK-like TCS and TCS-9 as they may represent new regulatory systems than can be involved in S. mutans pathogenesis.
Significance and Impact of the Study
Multiple TCS govern important biological parameters of S. mutans enabling its survival and persistence in the biofilm community.
acid resistance; biofilm formation; gene inactivation; oral streptococci; stress tolerance; two-component system
Genetic competence appears to be important in establishment of biofilms and tolerance of environmental insults. We report here that the development of competence is controlled at multiple levels in a complex network that includes two signal-transducing two-component systems (TCS). Using Streptococcus mutans strain UA159, we demonstrate that the histidine kinase CiaH, but not the response regulator CiaR, causes a dramatic decrease in biofilm formation and in transformation efficiency. Inactivation of comE or comD had no effect on stress tolerance, but transformability of the mutants was poor and was not restored by addition of competence-stimulating peptide (CSP). Horse serum (HS) or bovine serum albumin (BSA) had no impact on transformability of any strains. Interestingly, though, the presence of HS or BSA in combination with CSP was required for efficient induction of comD, comX, and comYA, and induction was dependent on ComDE and CiaH, but not CiaR. Inactivation of comC, encoding CSP, had no impact on transformation, and CiaH was shown to be required for optimal comC expression. This study reveals that S. mutans integrates multiple environmental signals through CiaHR and ComDE to coordinate induction of com genes and that CiaH can exert its influence through CiaR and as-yet-unidentified regulators. The results highlight critical differences in the role and regulation of CiaRH and com genes in different S. mutans isolates and between S. mutans and Streptococcus pneumoniae, indicating that substantial divergence in the role and regulation of TCS and competence genes has occurred in streptococci.
Two-component signal transduction systems (TCSs) in prokaryotes often regulate gene clusters that induce pathogenicity, and thus they have frequently been proposed as potential drug targets for attenuating the virulence of pathogens. The pathogenic potential of Streptococcus mutans, the major etiological pathogen of dental caries, is also regulated by its TCSs. The object of this study was to evaluate the effect of a histidine kinase (HK) inhibitor against two major virulence factors of S. mutans: biofilm formation and acid tolerance. Walkmycin C (WKM C), an HK inhibitor isolated from the screening of inhibitors against WalK HK in Bacillus subtilis, inhibited the in vitro autophosphorylation activity of three purified S. mutans HKs, i.e., VicK, CiaH, and LiaS. Although S. mutans does not have any essential HK but only an essential response regulator, VicR, WKM C showed an MIC of 6.25 μg/ml. This inhibitory effect of WKM C suggests that blocking the autophosphorylation of multiple HKs may inhibit phosphotransfer to VicR from VicK and other HKs. When WKM C was added at sub-MIC levels, the cells formed abnormal biofilms and also showed a defect in competence. When the cells were pretreated with WKM C, an increase in acid sensitivity was observed. Our results show that WKM C represses two pathogenic phenotypes of S. mutans, indicating the possibility of developing histidine kinase inhibitors into antivirulence drugs.
In the Streptococcus pneumoniae genome, stkP, encoding a membrane-associated serine/threonine kinase, is not redundant (L. Novakova, S. Romao, J. Echenique, P. Branny, and M.-C. Trombe, unpublished results). The data presented here demonstrate that StkP belongs to the signaling network involved in competence triggering in vitro and lung infection and bloodstream invasion in vivo. In competence, functional StkP is required for activation of comCDE upstream of the autoregulated ring orchestrated by the competence-stimulating peptide. This is the first description of positive regulation of comCDE transcription in balance with its repression by CiaRH.
In Streptococcus pneumoniae, a fermentative aerotolerant and catalase-deficient human pathogen, oxidases with molecular oxygen as substrate are important for virulence and for competence. The signal-transducing two-component systems CiaRH and ComDE mediate the response to oxygen, culminating in competence. In this work we show that the two-component MicAB system, whose MicB kinase carries a PAS domain, is also involved in competence repression under oxygen limitation. Autophosphorylation of recombinant MicB and phosphotransfer to recombinant MicA have been demonstrated. Mutational analysis and in vitro assays showed that the C-terminal part of the protein and residue L100 in the N-terminal cap of its PAS domain are both crucial for autokinase activity in vitro. Although no insertion mutation in micA was obtained, expression of the mutated allele micA59DA did not change bacterial growth and overcame competence repression under microaerobiosis. This was related to a strong instability of MicA59DA-PO4 in vitro. Thus, mutations which either reduced the stability of MicA-PO4 or abolished kinase activity in MicB were related to competence derepression under microaerobiosis, suggesting that MicA-PO4 is involved in competence repression when oxygen becomes limiting. The micAB genes are flanked by mutY and orfC. MutY is an adenine glycosylase involved in the repair of oxidized pyrimidines. OrfC shows the features of a metal binding protein. We did not obtain insertion mutation in orfC, suggesting its requirement for growth. It is proposed that MicAB, with its PAS motif, may belong to a set of functions important in the protection of the cell against oxidative stress, including the control of competence.
Nasopharyngeal carriage is the reservoir from which most disease with Streptococcus pneumoniae arises. Survival as a commensal in this environment is likely to require a set of adaptations distinct from those needed to cause disease, some of which may be mediated by two-component signal transduction systems (TCSTS). We examined the contributions of nine pneumococcal TCSTS to the process of nasopharyngeal colonization by using an infant rat model. Whereas deletions in all but one of these systems have been associated previously with a high degree of attenuation in a murine model of pneumonia, only the CiaRH system was necessary for efficient carriage. Transcriptional analysis by using microarray hybridization identified a locus consisting of two adjacent genes, htrA and spoJ, that was specifically and strongly downregulated in a ΔciaRH-null mutant. A S. pneumoniae strain lacking the htrA gene encoding a putative serine protease, but not one lacking spoJ, showed decreased fitness in a competitive model of colonization, a finding consistent with this gene mediating a portion of the carriage deficit observed with the ΔciaRH strain.
The Streptococcus pneumoniae ComDE two-component signaling system controls the development of genetic competence in the bacterium and affects virulence in models of pneumonia and bacteremia. We have investigated the impact of the competence pathway during colonization of the nasopharynx, the principal ecological niche of the pneumococcus. Previous work showed that deletion of the pneumococcal CiaRH signaling system inhibited colonization and increased expression of genes required for competence. We anticipated that signaling by the competence pathway might similarly reduce carriage. Consistent with this expectation, a comE deletion that blocked transformation increased colonization fitness such that the mutant outcompeted the wild type in an infant rat model of asymptomatic carriage. Deletion of comD—immediately upstream of comE and likewise required for competence—similarly increased colonization fitness if the orientation of the antibiotic resistance cassette inserted into the comD locus was such that it reduced transcription of comE. However, an alternative comD deletion mutation that caused an increase in comE transcription impaired colonization instead. Activation of the competence system through a comE(D143Y) mutation did not affect colonization, but an inability to secrete the competence-stimulating peptide due to deletion of comAB produced a density-dependent reduction in colonization fitness. These results suggest a model in which signaling by the unactivated form of ComE reduces colonization fitness compared to that of bacteria in which it is either activated or absent entirely, with the most substantial fitness gain accompanying deletion of comE. This observation demonstrates that the pneumococcus incurs a substantial fitness cost in order to retain a functional competence regulatory system.
The homolactic and catalase-deficient pathogen Streptococcus pneumoniae is not only tolerant to oxygen but requires the activity of its NADH oxidase, Nox, to develop optimal virulence and competence for genetic transformation. In this work, we show that the global regulator RegR is also involved in these traits. Genetic dissection revealed that RegR regulates competence and the expression of virulence factors, including hyaluronidase. In bacteria grown in vitro, RegR represses hyaluronidase. At neutral pH, it increases adherence to A549 epithelial cells, and at alkaline pH, it acts upstream of the CiaRH two-component signaling system to activate competence. These phenotypes are not associated with changes in antibiotic resistance, central metabolism, and carbohydrate utilization. Although the RegR0 (where 0 indicates the loss of the protein) mutation is sufficient to attenuate experimental virulence of strain 23477 in mice, the introduction of an additional hyl0 (where 0 indicates the loss of function) mutation in the RegR0 strain 23302 dramatically reduces its virulence. This indicates that residual virulence of the RegR0 Hyl+ derivative is due to hyaluronidase and supports the dual role of RegR in virulence. This LacI/GalR regulator, not essential for in vitro growth in rich media, is indeed involved in the adaptive response of the pneumococcus via its control of competence, adherence, and virulence.
Group B Streptococcus (GBS) is major cause of invasive disease in newborn infants and the leading cause of neonatal meningitis. To gain access to the central nervous system (CNS), GBS must not only subvert host defenses in the bloodstream but also invade and survive within brain microvascular endothelial cells (BMEC), the principal cell layer composing the blood-brain barrier (BBB). While several GBS determinants that contribute to the invasion of BMEC have been identified, little is known about the GBS factors that are required for intracellular survival and ultimate disease progression. In this study we sought to identify these factors by screening a random GBS mutant library in an in vitro survival assay. One mutant was identified which contained a disruption in a two-component regulatory system homologous to CiaR/CiaH, which is present in other streptococcal pathogens. Deletion of the putative response regulator, ciaR, in GBS resulted in a significant decrease in intracellular survival within neutrophils, murine macrophages, and human BMEC, which was linked to increased susceptibility to killing by antimicrobial peptides, lysozyme, and reactive oxygen species. Furthermore, competition experiments with mice showed that wild-type GBS had a significant survival advantage over the GBS ΔciaR mutant in the bloodstream and brain. Microarray analysis comparing gene expression between wild-type and ΔciaR mutant GBS bacteria revealed several CiaR-regulated genes that may contribute to stress tolerance and the subversion of host defenses by GBS. Our results identify the GBS CiaR response regulator as a crucial factor in GBS intracellular survival and invasive disease pathogenesis.
The CiaR/H two-component system is involved in regulating virulence and competence in Streptococcus pneumoniae. The system is known to regulate many genes, including that for high-temperature requirement A (HtrA). This gene has been implicated in the ability of the pneumococcus to colonize the nasopharynx of infant rats. We reported previously that deletion of the gene for HtrA made the pneumococcal strains much less virulent in mouse models, less able to grow at higher temperatures, and more sensitive to oxidative stress. In this report, we show that the growth phenotype as well as sensitivity to oxidative stress of ΔciaR mutant was very similar to that of a ΔhtrA mutant and that the expression of the HtrA protein was reduced in a ciaR-null mutant. Both the in vitro phenotype and the reduced virulence of ΔciaR mutant could be restored by increasing the expression of HtrA.
Induction of the agmatine deiminase system (AgDS) of Streptococcus mutans requires agmatine and is optimal at low pH. We show here that the VicRK, ComDE, and CiaRH two-component systems influence AgDS gene expression in response to acidic and thermal stresses.
Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Acute C. jejuni-mediated disease (campylobacteriosis) involves C. jejuni invasion of host epithelial cells using adhesins (e.g., CadF and FlpA) and secreted proteins [e.g., the Campylobacter invasion antigens (Cia)]. The genes encoding the Cia proteins are up-regulated upon co-culture of C. jejuni with epithelial cells. One of the Cia proteins, CiaC, is required for maximal invasion of host cells by C. jejuni. Previous work has also revealed that CiaC is, in part, responsible for host cell cytoskeletal rearrangements that result in membrane ruffling. This study was performed to test the hypothesis that CiaC is delivered to the cytosol of host cells. To detect the delivery of CiaC into cultured epithelial cells, we used the adenylate cyclase domain (ACD) of Bordetella pertussis CyaA as a reporter. In this study, we found that export and delivery of the C. jejuni Cia proteins into human INT 407 epithelial cells required a functional flagellar hook complex composed of FlgE, FlgK, and FlgL. Assays performed with bacterial culture supernatants supported the hypothesis that CiaC delivery requires bacteria-host cell contact. We also found that CiaC was delivered to host cells by cell-associated (bound) bacteria, as judged by experiments performed with inhibitors that specifically target the cell signaling pathways utilized by C. jejuni for cell invasion. Interestingly, the C. jejuni flgL mutant, which is incapable of exporting and delivering the Cia proteins, did not induce INT 407 cell membrane ruffles. Complementation of the flgL mutant with plasmid-encoded flgL restored the motility and membrane ruffling. These data support the hypothesis that the C. jejuni Cia proteins, which are exported from the flagellum, are delivered to the cytosol of host cells.
T3SS; flagellum; effector proteins; adenylate cyclase; membrane ruffling
The vancomycin stress response was studied in Streptococcus pneumoniae strains T4 (TIGR4) and Tupelo. Vancomycin affected the expression of 175 genes, including genes encoding transport functions and enzymes involved in aminosugar metabolism. The two-component systems TCS03, TCS11, and CiaRH also responded to antibiotic treatment. We hypothesize that the three regulons are an important part of the bacterium's response to vancomycin stress.
The oral commensal Streptococcus gordonii must adapt to constantly fluctuating and often hostile environmental conditions to persist in the oral cavity. The arginine deiminase system (ADS) of S. gordonii enables cells to produce, ornithine, ammonia, CO2, and ATP from arginine hydrolysis, augmenting the acid tolerance of the organism. The ADS genes are substrate inducible and sensitive to catabolite repression, mediated through ArcR and CcpA, respectively, but the system also requires low pH and anaerobic conditions for optimal activation. Here, we demonstrate that the CiaRH and ComDE two-component systems (TCS) are required for low-pH-dependent expression of ADS genes in S. gordonii. Further, the VicRK TCS is required for optimal ADS gene expression under anaerobic conditions and enhances the sensitivity of the operon to repression by oxygen. The known anaerobic activator of the ADS, Fnr-like protein (Flp), appeared to act independently of the Vic TCS. Mutants of S. gordonii lacking components of the CiaRH, ComDE, or VicRK grew more slowly in acidified media and were more sensitive to killing at lethal pH values and to agents that induce oxidative stress. This study provides the first evidence that TCS can regulate the ADS of bacteria in response to specific environmental signals and reveals some notable differences in the contribution of CiaRH, ComDE, and VicRK to viability and stress tolerance between the oral commensal S. gordonii and the oral pathogen Streptococcus mutans.
The two-component system TCS08 is one of the regulatory systems that is important for virulence of Streptococcus pneumoniae. In order to investigate the TCS08 regulon, we have analyzed transcription profiles of mutants derived from S. pneumoniae R6 by microarray analysis. Since deletion mutants are often without a significant phenotype, we constructed a mutation in the histidine kinase HK08, T133P, in analogy to the phosphatase mutation T230P in the H box of the S. pneumoniae CiaH kinase described recently (D. Zähner, K. Kaminski, M. van der Linden, T. Mascher, M. Merai, and R. Hakenbeck, J. Mol. Microbiol. Biotechnol. 4:211-216, 2002). In addition, a deletion mutation was constructed in rr08, encoding the cognate response regulator. The most heavily suppressed genes in the hk08 mutant were spr0276 to spr0282, encoding a putative cellobiose phosphoenolpyruvate sugar phosphotransferase system (PTS). Whereas the R6 Smr parent strain and the Δrr08 mutant readily grew on cellobiose, the hk08 mutant and selected mutants with deletions in the PTS cluster did not, strongly suggesting that TCS08 is involved in the catabolism of cellobiose. Homologues of the TCS08 system were found in closely related streptococci and other gram-positive cocci. However, the genes spr0276 to spr0282, encoding the putative cellobiose PTS, represent a genomic island in S. pneumoniae and homologues were found in Streptococcus gordonii only, suggesting that this system might contribute to the pathogenicity potential of the pneumococcus.
Many clinical isolates of Streptococcus mutans produce peptide antibiotics called mutacins. Mutacin production may play an important role in the ecology of S. mutans in dental plaque. In this study, inactivation of a histidine kinase gene, ciaH, abolished mutacin production. Surprisingly, the same mutation also diminished competence development, stress tolerance, and sucrose-dependent biofilm formation.
Penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae represents a primary resistance determinant for beta-lactams, and low-affinity PBP2x variants can easily be selected with cefotaxime. Penicillin-resistant clinical isolates of S. pneumoniae frequently contain in their mosaic PBP2x the mutation T338A adjacent to the active site S337, and T338P as well as T338G substitutions are also known. Site-directed mutagenesis has now documented that a single point mutation at position T338 confers selectable levels of beta-lactam resistance preferentially to oxacillin. Despite the moderate impact on beta-lactam susceptibility, the function of the PBP2x mutants appears to be impaired, as can be documented in the absence of a functional CiaRH regulatory system, resulting in growth defects and morphological changes. The combination of low-affinity PBP2x and PBP1a encoded by mosaic genes is known to result in high cefotaxime resistance. In contrast, introduction of a mosaic pbp1a into the PBP2xT338G mutant did not lead to increased resistance. However, the mosaic PBP1a gene apparently complemented the PBP2xT338G defect, since Cia mutant derivatives grew normally. The data support the view that PBP2x and PBP1a interact with each other on some level and that alterations of both PBPs in resistant clinical isolates have evolved to ensure cooperation between both proteins.
Cia3 is a locus on rat chromosome 4 that regulates severity and joint damage in collagen and pristane-induced arthritis (CIA and PIA). This study aimed to refine the Cia3 gene-containing interval towards gene identification and obtain insights into its mode of action.
Five DA.F344(Cia3) subcongenic strains were generated and studied in PIA and CIA. Levels of antibodies against type II collagen (both allo- and autoantibodies) were measured. Joints and synovial tissues were collected 32 days after the induction of PIA (chronic stage) for histology and qPCR for IL-1β and matrix metalloproteases (MMPs).
Three subcongenics sharing the centromeric Cia3d interval were protected, while two subcongenics sharing the telomeric Cia3g interval, which did not overlap with Cia3d, were also protected, developing significantly less severe CIA and PIA. DA.F344(Cia3) and DA.F344(Cia3d) congenics with PIA preserved a normal joint architecture, while DA rats had pronounced synovial hyperplasia, angiogenesis, inflammatory infiltration, bone or cartilage erosions. DA.F344(Cia3d) and DA.F344(Cia3g) strains had significantly lower synovial levels of IL-1β (5-fold), MMP-1 (expressed predominantly in DA), MMP-3 (79-fold) and MMP-14 (21-fold) and reduced levels of pathogenic autoantibodies against type II collagen, compared with DA.
We have identified two new arthritis severity and articular damage loci within Cia3. These loci regulate pathogenic processes in two different models of RA, and the identification of these genes has the potential to generate new targets for therapies aimed at reducing disease severity and articular damage, and for prognostication in RA.
Autoimmunity; Rheumatoid arthritis; animal model; erosion; IL-1β
Autoimmune diseases, like rheumatoid arthritis, result from a dysregulation of the immune response culminating in hyperactivation of effector cells leading to immune-mediated injury. To maintain an appropriate immune response and prevent the emergence of autoimmune disease, activation signals must be regulated by inhibitory pathways. Biochemical and genetic studies indicate that the type IIB low-affinity receptor for immunoglobulin (Ig)G (FcγRIIB) inhibits cellular activation triggered through antibody or immune complexes and may be an important component in preventing the emergence of autoimmunity. To investigate the role of FcγRIIB in the development of type II collagen (CII)-induced arthritis (CIA), a model for rheumatoid arthritis in humans, we have examined its contribution in determining the susceptibility to CIA in the nonpermissive H-2b haplotype. H-2b mice immunized with bovine CII do not develop appreciable disease. In contrast, immunization of the FcγRIIB-deficient, H-2b mice with bovine CII induced CIA at an incidence of 42.2%. The maximal arthritis index of the FcγRIIB-deficient mice developing CIA (6.9 ± 3.6) was comparable to that of DBA/1 mice (8.6 ± 1.9), an H-2q strain susceptible for CIA induction. IgG1, IgG2a, and IgG2b antibody responses against CII were elevated in the FcγRIIB-deficient animals, especially in those mice showing arthritis, but less pronounced than DBA/1 mice. Histological examinations of the arthritic paws from FcγRIIB-deficient mice revealed that cartilage was destroyed and bone was focally eroded in association with marked lymphocyte and monocyte/macrophage infiltration, very similar to the pathologic findings observed in DBA/1 mice. These results indicate that a nonpermissive H-2b haplotype can be rendered permissive to CIA induction through deletion of FcγRIIB, suggesting that FcγRIIB plays a critical role in suppressing the induction of CIA.
collagen-induced arthritis; autoimmunity; Fc receptor; gene targeting; macrophage