The emergence and diversification of a zoonotic pathogen from within the microbiota of intensively farmed pigs.

The expansion and intensification of livestock production is predicted to promote the emergence of pathogens. As pathogens sometimes jump between species, this can affect the health of humans as well as livestock. Here, we investigate how livestock microbiota can act as a source of these emerging pathogens through analysis of Streptococcus suis, a ubiquitous component of the respiratory microbiota of pigs that is also a major cause of disease on pig farms and an important zoonotic pathogen. Combining molecular dating, phylogeography, and comparative genomic analyses of a large collection of isolates, we find that several pathogenic lineages of S. suis emerged in the 19th and 20th centuries, during an early period of growth in pig farming. These lineages have since spread between countries and continents, mirroring trade in live pigs. They are distinguished by the presence of three genomic islands with putative roles in metabolism and cell adhesion, and an ongoing reduction in genome size, which may reflect their recent shift to a more pathogenic ecology. Reconstructions of the evolutionary histories of these islands reveal constraints on pathogen emergence that could inform control strategies, with pathogenic lineages consistently emerging from one subpopulation of S. suis and acquiring genes through horizontal transfer from other pathogenic lineages. These results shed light on the capacity of the microbiota to rapidly evolve to exploit changes in their host population and suggest that the impact of changes in farming on the pathogenicity and zoonotic potential of S. suis is yet to be fully realized.

Sero- and apx-typing of German Actinobacillus pleuropneumoniae field isolates from 2010 to 2019 reveals a predominance of serovar 2 with regular apx-profile.

Serotyping is the most common method to characterize field isolates of Actinobacillus (A.) pleuropneumoniae, the etiological agent of porcine pleuropneumonia. Based on serology, many farms seem to be infected and antibodies against a wide variety of serovars are detectable, but, so far it is unknown to what degree respective serovars contribute to outbreaks of clinical manifest disease. In this study, 213 German A. pleuropneumoniae field isolates retrieved for diagnostic purposes from outbreaks of porcine pleuropneumonia between 2010 and 2019 were genetically serotyped and analyzed regarding their apx-toxin gene profile using molecular methods. Serotyping revealed a prominent role of serovar 2 in clinical cases (64% of all isolates) and an increase in the detection of this serovar since 2010 in German isolates. Serovar 9/11 followed as the second most frequent serovar with about 15% of the isolates. Furthermore, very recently described serovars 16 (n = 2) and 18 (n = 8) were detected. Most isolates (93.4%) showed apx-profiles typical for the respective serovar. However, this does not hold true for isolates of serovar 18, as 75% (n = 6) of all isolates of this serovar deviated uniformly from the "typical" apx-gene profile of the reference strain 7311555. Notably, isolates from systemic lesions such as joints or meninges did not harbor the complete apxICABD operon which is considered typical for highly virulent strains. Furthermore, the extremely low occurrence (n = 1) of NAD independent (biovar II) isolates in German A. pleuropneumoniae was evident in our collection of clinical isolates.

Immunogenicity and protective efficacy of a Streptococcus suis vaccine composed of six conserved immunogens.

A vaccine protecting against different Streptococcus suis serotypes is highly needed in porcine practice to improve animal welfare and reduce the use of antibiotics. We hypothesized that immunogens prominently recognized by convalescence sera but significantly less so by sera of susceptible piglets are putative protective antigens. Accordingly, we investigated immunogenicity and protective efficacy of a multicomponent vaccine including six main conserved immunogens, namely SSU0934, SSU1869, SSU0757, SSU1950, SSU1664 and SSU0187. Flow cytometry confirmed surface expression of all six immunogens in S. suis serotypes 2, 9 and 14. Although prime-booster vaccination after weaning resulted in significantly higher specific IgG levels against all six immunogens compared to the placebo-treated group, no significant differences between bacterial survival in blood from either vaccinated or control animals were recorded for serotype 2, 9 and 14 strains. Furthermore, vaccinated piglets were not protected against morbidity elicited through intranasal challenge with S. suis serotype 14. As ~50% of animals in both groups did not develop disease, we investigated putative other correlates of protection. Induction of reactive oxygen species (ROS) in blood granulocytes was not associated with vaccination but correlated with protection as all piglets with >5% ROS survived the challenge. Based on these findings we discuss that the main immunogens of S. suis might actually not be a priori good candidates for protective antigens. On the contrary, expression of immunogens that evoke antibodies that do not mediate killing of this pathogen might constitute an evolutionary advantage conserved in many different S. suis strains.

Candidate genes and gene markers for the resistance to porcine pleuropneumonia.

Actinobacillus (A.) pleuropneumoniae is one of the most important respiratory pathogens in global pig production. Antimicrobial treatment and vaccination provide only limited protection, but genetic disease resistance is a very promising alternative for sustainable prophylaxis. Previous studies have discovered multiple QTL that may explain up to 30% of phenotypic variance. Based on these findings, the aim of the present study was to use genomic sequencing to identify genetic markers for resistance to pleuropneumonia in a segregating commercial German Landrace line. 163 pigs were infected with A. pleuropneumoniae Serotype 7 through a standardized aerosol infection method. Phenotypes were accurately defined on a clinical, pathological and microbiological basis. The 58 pigs with the most extreme phenotypes were genotyped by sequencing (next-generation sequencing). SNPs were used in a genome-wide association study. The study identified genome-wide associated SNPs on three chromosomes, two of which were chromosomes of QTL which had been mapped in a recent experiment. Each variant explained up to 20% of the total phenotypic variance. Combined, the three variants explained 52.8% of the variance. The SNPs are located in genes involved in the pathomechanism of pleuropneumonia. This study confirms the genetic background for the host's resistance to pleuropneumonia and indicates a potential role of three candidates on SSC2, SSC12 and SSC15. Favorable gene variants are segregating in commercial populations. Further work is needed to verify the results in a controlled study and to identify the functional QTN.

Serotyping and pathotyping of Glaesserella parasuis isolated 2012-2019 in Germany comparing different PCR-based methods.

Glaesserella parasuis is an important pathogen in swine production. It acts as a primary pathogen in systemic Glässer´s disease and as a secondary pathogen in Porcine Respiratory Disease Complex. In this study, a collection of 308 isolates from carrier animals and individuals with respiratory or Glässer´s disease isolated 2012-2019 in Germany was analysed. Isolates were characterized for serovar implementing two different PCR methods. Additionally, two different PCR methods for pathotyping isolates were applied to the collection and results compared. Serovar 6 (p < 0.0001) and 9 (p = 0.0007) were correlated with carrier isolates and serovar 4 was associated with isolates from animals with respiratory disease (p = 0.015). In systemic isolates, serovar 13 was most frequently detected (18.9%). Various other serovars were isolated from all sites and the ratio of serovar 5 to serovar 12 was approximately 1:2. These two serovars together represented 14.3% of the isolates; only serovar 4 was isolated more frequently (24.7%). The pathotyping method based on the leader sequence (LS = ESPR of vta) was easy to perform and corresponded well to the clinical background information. Of the carrier isolates 72% were identified as non-virulent while 91% of the systemic isolates were classified as virulent (p < 0.0001). Results of the pathotyping PCR based on 10 different marker genes overall were in good agreement with clinical metadata as well as with results of the LS-PCR. However, the pathotyping PCR was more complicated to perform and analyze. In conclusion, a combination of the serotyping multiplex-PCR and the LS-PCR could improve identification of clinically relevant G. parasuis isolates, especially from respiratory samples.

Infection of bovine well-differentiated airway epithelial cells by Pasteurella multocida: actions and counteractions in the bacteria-host interactions.

Pasteurella (P.) multocida is a zoonotic pathogen, which is able to cause respiratory disorder in different hosts. In cattle, P. multocida is an important microorganism involved in the bovine respiratory disease complex (BRDC) with a huge economic impact. We applied air-liquid interface (ALI) cultures of well-differentiated bovine airway epithelial cells to analyze the interaction of P. multocida with its host target cells. The bacterial pathogen grew readily on the ALI cultures. Infection resulted in a substantial loss of ciliated cells. Nevertheless, the epithelial cell layer maintained its barrier function as indicated by the transepithelial electrical resistance and the inability of dextran to get from the apical to the basolateral compartment via the paracellular route. Analysis by confocal immunofluorescence microscopy confirmed the intactness of the epithelial cell layer though it was not as thick as the uninfected control cells. Finally, we chose the bacterial neuraminidase to show that our infection model is a sustainable tool to analyze virulence factors of P. multocida. Furthermore, we provide an explanation, why this microorganism usually is a commensal and becomes pathogenic only in combination with other factors such as co-infecting microorganisms.

Role of Bacterial and Host DNases on Host-Pathogen Interaction during Streptococcus suis Meningitis.

Streptococcus suis is a zoonotic agent causing meningitis in pigs and humans. Neutrophils, as the first line of defense against S. suis infections, release neutrophil extracellular traps (NETs) to entrap pathogens. In this study, we investigated the role of the secreted nuclease A of S. suis (SsnA) as a NET-evasion factor in vivo and in vitro. Piglets were intranasally infected with S. suis strain 10 or an isogenic ssnA mutant. DNase and NET-formation were analyzed in cerebrospinal fluid (CSF) and brain tissue. Animals infected with S. suis strain 10 or S. suis 10ΔssnA showed the presence of NETs in CSF and developed similar clinical signs. Therefore, SsnA does not seem to be a crucial virulence factor that contributes to the development of meningitis in pigs. Importantly, DNase activity was detectable in the CSF of both infection groups, indicating that host nucleases, in contrast to bacterial nuclease SsnA, may play a major role during the onset of meningitis. The effect of DNase 1 on neutrophil functions was further analyzed in a 3D-cell culture model of the porcine blood-CSF barrier. We found that DNase 1 partially contributes to enhanced killing of S. suis by neutrophils, especially when plasma is present. In summary, host nucleases may partially contribute to efficient innate immune response in the CSF.

Viral Coinfection Replaces Effects of Suilysin on Streptococcus suis Adherence to and Invasion of Respiratory Epithelial Cells Grown under Air-Liquid Interface Conditions.

Streptococcus suis is an important zoonotic pathogen which can infect humans and pigs worldwide, posing a potential risk to global public health. Suilysin, a pore-forming cholesterol-dependent cytolysin, is considered to play an important role in the pathogenesis of S. suis infections. It is known that infection with influenza A viruses may favor susceptibility to secondary bacterial infection, resulting in more severe disease and increased mortality. However, the molecular mechanisms underlying these coinfections are incompletely understood. Applying highly differentiated primary porcine respiratory epithelial cells grown under air-liquid interface (ALI) conditions, we analyzed the contribution of swine influenza viruses (SIV) to the virulence of S. suis, with a special focus on its cytolytic toxin, suilysin. We found that during secondary bacterial infection, suilysin of S. suis contributed to the damage of well-differentiated respiratory epithelial cells in the early stage of infection, whereas the cytotoxic effects induced by SIV became prominent at later stages of infection. Prior infection by SIV enhanced the adherence to and colonization of porcine airway epithelial cells by a wild-type (wt) S. suis strain and a suilysin-negative S. suis mutant in a sialic acid-dependent manner. A striking difference was observed with respect to bacterial invasion. After bacterial monoinfection, only the wt S. suis strain showed an invasive phenotype, whereas the mutant remained adherent. When the epithelial cells were preinfected with SIV, the suilysin-negative mutant also showed an invasion capacity. Therefore, we propose that coinfection with SIV may compensate for the lack of suilysin in the adherence and invasion process of suilysin-negative S. suis.

Link between Heterotrophic Carbon Fixation and Virulence in the Porcine Lung Pathogen Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a capnophilic pathogen of the porcine respiratory tract lacking enzymes of the oxidative branch of the tricarboxylic acid (TCA) cycle. We previously claimed that A. pleuropneumoniae instead uses the reductive branch in order to generate energy and metabolites. Here, we show that bicarbonate and oxaloacetate supported anaerobic growth of A. pleuropneumoniae Isotope mass spectrometry revealed heterotrophic fixation of carbon from stable isotope-labeled bicarbonate by A. pleuropneumoniae, which was confirmed by nano-scale secondary ion mass spectrometry at a single-cell level. By gas chromatography-combustion-isotope ratio mass spectrometry we could further show that the labeled carbon atom is mainly incorporated into the amino acids aspartate and lysine, which are derived from the TCA metabolite oxaloacetate. We therefore suggest that carbon fixation occurs at the interface of glycolysis and the reductive branch of the TCA cycle. The heme precursor δ-aminolevulinic acid supported growth of A. pleuropneumoniae, similar to bicarbonate, implying that anaplerotic carbon fixation is needed for heme synthesis. However, deletion of potential carbon-fixing enzymes, including PEP-carboxylase (PEPC), PEP-carboxykinase (PEPCK), malic enzyme, and oxaloacetate decarboxylase, as well as various combinations thereof, did not affect carbon fixation. Interestingly, generation of a deletion mutant lacking all four enzymes was not possible, suggesting that carbon fixation in A. pleuropneumoniae is an essential metabolic pathway controlled by a redundant set of enzymes. A double deletion mutant lacking PEPC and PEPCK was not impaired in carbon fixation in vitro but showed reduction of virulence in a pig infection model.

Highly Pathogenic Avian Influenza A(H5N8) Virus in Gray Seals, Baltic Sea.

We detected a highly pathogenic avian influenza A(H5N8) virus in lung samples of 2 gray seals (Halichoerus grypus) stranded on the Baltic coast of Poland in 2016 and 2017. This virus, clade 2.3.4.4 B, was closely related to avian H5N8 viruses circulating in Europe at the time.

Sialic acid-dependent interaction of group B streptococci with influenza virus-infected cells reveals a novel adherence and invasion mechanism.

Group B streptococci (GBS) contain a capsular polysaccharide with side chains terminating in α2,3-linked sialic acids. Because of this linkage type, the sialic acids of GBS are recognised by lectins of immune cells. This interaction results in a dampening of the host immune response and thus promotes immune evasion. As several influenza A viruses (IAV) use α2,3-linked sialic acid as a receptor determinant for binding to host cells, we analysed whether GBS and influenza viruses can interact with each other and how this interaction affects viral replication and bacterial adherence to and invasion of host cells. A co-sedimentation assay revealed that viruses with a preference for α2,3-linked sialic acids bind to GBS in a sialic acid-dependent manner. There is, however, a large variation in the efficiency of binding among avian influenza viruses of different subtypes as shown by a hemagglutination-inhibition assay. A delay in the growth curve of IAV indicated that GBS has an inhibitory effect on virus replication. On the other hand, both the adherence and invasion efficiency of GBS were enhanced when the cells were pre-infected by IAV with appropriate receptor specificity. Our results suggest that GBS infection may result in a more severe disease when patients are co-infected by influenza viruses. This co-infection mechanism may have relevance also to other human diseases, as there are more bacterial pathogens with α2,3-linked sialic acids and human viruses binding to this linkage type.

Neutrophil extracellular trap formation in the Streptococcus suis-infected cerebrospinal fluid compartment.

Streptococcus suis is an important meningitis-causing pathogen in pigs and humans. Neutrophil extracellular traps (NETs) have been identified as host defense mechanism against different pathogens. Here, NETs were detected in the cerebrospinal fluid (CSF) of S. suis-infected piglets despite the presence of active nucleases. To study NET-formation and NET-degradation after transmigration of S. suis and neutrophils through the choroid plexus epithelial cell barrier, a previously described model of the human blood-CSF barrier was used. NETs and respective entrapment of streptococci were recorded in the "CSF compartment" despite the presence of active nucleases. Comparative analysis of S. suis wildtype and different S. suis nuclease mutants did not reveal significant differences in NET-formation or bacterial survival. Interestingly, transcript expression of the human cathelicidin LL-37, a NET-stabilizing factor, increased after transmigration of neutrophils through the choroid plexus epithelial cell barrier. In good accordance, the porcine cathelicidin PR-39 was significantly increased in CSF of piglets with meningitis. Furthermore, we confirmed that PR-39 is associated with NETs in infected CSF and inhibits neutrophil DNA degradation by bacterial nucleases. In conclusion, neutrophils form NETs after breaching the infected choroid plexus epithelium, and those NETs may be protected by antimicrobial peptides against bacterial nucleases.

Multi-organ spreading of Actinobacillus pleuropneumoniae serovar 7 in weaned pigs during the first week after experimental infection.

Actinobacillus (A.) pleuropneumoniae is normally considered strictly adapted to the respiratory tract of swine. Despite this, scattered case reports of arthritis, osteomyelitis, hepatitis, meningitis or nephritis exist, in which A. pleuropneumoniae remained the only detectable pathogen. Therefore, the aim of this study was to investigate whether spreading to other organs than the lungs is incidental or may occur more frequently. For this, organ samples (blood, liver, spleen, kidney, tarsal and carpal joints, meninges, pleural and pericardial fluids) from weaners (n = 47) infected experimentally with A. pleuropneumoniae serovar 7 by aerosol infection (infection dose: 10.9 × 103 cfu/animal) were examined by culture during the first week after infection. In addition, tissue samples of eight weaners were examined by histology and immunohistochemistry (IHC). A. pleuropneumoniae was isolated in all examined sample sites (86.7% pleural fluids, 73.3% pericardial fluids, 50.0% blood, 61.7% liver, 51.1% spleen, 55.3% kidney, 14.9% tarsal joints, 12.8% carpal joints, 27.7% meninges). These results were also obtained from animals with only mild clinical symptoms. IHC detection confirmed these findings in all locations except carpal joints. Histological examination revealed purulent hepatitis (n = 2), nephritis (n = 1) and beginning meningitis (n = 2). Isolation results were significantly correlated (p < 0.001) with the degree of lung colonization and, to a lower extent, with the severity of disease. Detection of A. pleuropneumoniae in peripheral tissues was significantly correlated to spleen colonization. In conclusion, multi-organ spreading of A. pleuropneumoniae serovar 7 strain AP 76 seems to occur more frequently during acute infection following effective lung colonization than previously thought.

Characterization of the zinc metalloprotease of Streptococcus suis serotype 2.

Streptococcus suis is a swine pathogen and zoonotic agent responsible for meningitis and septic shock. Although several putative virulence factors have been described, the initial steps of the S. suis pathogenesis remain poorly understood. While controversial results have been reported for a S. suis serotype 2 zinc metalloprotease (Zmp) regarding its IgA protease activity, recent phylogenetic analyses suggested that this protein is homologous to the ZmpC of Streptococcus pneumoniae, which is not an IgA protease. Based on the previously described functions of metalloproteases (including IgA protease and ZmpC), different experiments were carried out to study the activities of that of S. suis serotype 2. First, results showed that S. suis, as well as the recombinant Zmp, were unable to cleave human IgA1, confirming lack of IgA protease activity. Similarly, S. suis was unable to cleave P-selectin glycoprotein ligand-1 and to activate matrix metalloprotease 9, at least under the conditions tested. However, S. suis was able to partially cleave mucin 16 and syndecan-1 ectodomains. Experiments carried out with an isogenic Δzmp mutant showed that the Zmp protein was partially involved in such activities. The absence of a functional Zmp protein did not affect the ability of S. suis to adhere to porcine bronchial epithelial cells in vitro, or to colonize the upper respiratory tract of pigs in vivo. Taken together, our results show that S. suis serotype 2 Zmp is not a critical virulence factor and highlight the importance of independently confirming results on S. suis virulence by different teams.

The inflammatory response and neuronal injury in Streptococcus suis meningitis.

BACKGROUND: Many of the currently used models of bacterial meningitis have limitations due to direct inoculation of pathogens into the cerebrospinal fluid or brain and a relatively insensitive assessment of long-term sequelae. The present study evaluates the utility of a Streptococcus (S.) suis intranasal infection model for the investigation of experimental therapies in meningitis. METHODS: We examined the brains of 10 piglets with S. suis meningitis as well as 14 control piglets by histology, immunohistochemistry and in-situ tailing for morphological alterations in the hippocampal dentate gyrus and microglial activation in the neocortex. RESULTS: In piglets with meningitis, the density of apoptotic neurons was significantly higher than in control piglets. Moreover, scoring of microglial morphology revealed a significant activation of these cells during meningitis. The slight increase in the density of dividing cells, young neurons and microglia observed in piglets suffering from meningitis was not statistically significant, probably because of the short time frame between onset of clinical signs and organ sampling. CONCLUSIONS: The morphological changes found during S. suis meningitis are in accordance with abnormalities in other animal models and human autopsy cases. Therefore, the pig should be considered as a model for evaluating effects of experimental therapeutic approaches on neurological function in bacterial meningitis.

Identification and Characterization of IgdE, a Novel IgG-degrading Protease of Streptococcus suis with Unique Specificity for Porcine IgG.

Streptococcus suisis a major endemic pathogen of pigs causing meningitis, arthritis, and other diseases. ZoonoticS. suisinfections are emerging in humans causing similar pathologies as well as severe conditions such as toxic shock-like syndrome. Recently, we discovered an IdeS family protease ofS. suisthat exclusively cleaves porcine IgM and represents the first virulence factor described, linkingS. suisto pigs as their natural host. Here we report the identification and characterization of a novel, unrelated protease ofS. suisthat exclusively targets porcine IgG. This enzyme, designated IgdE forimmunoglobulinG-degradingenzyme ofS. suis, is a cysteine protease distinct from previous characterized streptococcal immunoglobulin degrading proteases of the IdeS family and mediates efficient cleavage of the hinge region of porcine IgG with a high degree of specificity. The findings that allS. suisstrains investigated possess the IgG proteolytic activity and that piglet serum samples contain specific antibodies against IgdE strongly indicate that the protease is expressedin vivoduring infection and represents a novel and putative important bacterial virulence/colonization determinant, and a thus potential therapeutic target.

Use of Antibiotics and Antimicrobial Resistance in Veterinary Medicine as Exemplified by the Swine Pathogen Streptococcus suis.

Use of antimicrobial agents in veterinary medicine is essential to control infectious diseases, thereby keeping animals healthy and animal products safe for the consumer. On the other hand, development and spread of antimicrobial resistance is of major concern for public health. Streptococcus (S.) suis reflects a typical bacterial pathogen in modern swine production due to its facultative pathogenic nature and wide spread in the pig population. Thus, in the present review we focus on certain current aspects and problems related to antimicrobial use and resistance in S. suis as a paradigm for a bacterial pathogen affecting swine husbandry worldwide. The review includes (i) general aspects of antimicrobial use and resistance in veterinary medicine with emphasis on swine, (ii) genetic resistance mechanisms of S. suis known to contribute to bacterial survival under antibiotic selection pressure, and (iii) possible other factors which may contribute to problems in antimicrobial therapy of S. suis infections, such as bacterial persister cell formation, biofilm production, and co-infections. The latter shows that we hardly understand the complexity of factors affecting the success of antimicrobial treatment of (porcine) infectious diseases and underlines the need for further research in this field.

Characterization of the pivotal carbon metabolism of Streptococcus suis serotype 2 under ex vivo and chemically defined in vitro conditions by isotopologue profiling.

Streptococcus suis is a neglected zoonotic pathogen that has to adapt to the nutritional requirements in the different host niches encountered during infection and establishment of invasive diseases. To dissect the central metabolic activity of S. suis under different conditions of nutrient availability, we performed labeling experiments starting from [(13)C]glucose specimens and analyzed the resulting isotopologue patterns in amino acids of S. suis grown under in vitro and ex vivo conditions. In combination with classical growth experiments, we found that S. suis is auxotrophic for Arg, Gln/Glu, His, Leu, and Trp in chemically defined medium. De novo biosynthesis was shown for Ala, Asp, Ser, and Thr at high rates and for Gly, Lys, Phe, Tyr, and Val at moderate or low rates, respectively. Glucose degradation occurred mainly by glycolysis and to a minor extent by the pentose phosphate pathway. Furthermore, the exclusive formation of oxaloacetate by phosphoenolpyruvate (PEP) carboxylation became evident from the patterns in de novo synthesized amino acids. Labeling experiments with S. suis grown ex vivo in blood or cerebrospinal fluid reflected the metabolic adaptation to these host niches with different nutrient availability; however, similar key metabolic activities were identified under these conditions. This points at the robustness of the core metabolic pathways in S. suis during the infection process. The crucial role of PEP carboxylation for growth of S. suis in the host was supported by experiments with a PEP carboxylase-deficient mutant strain in blood and cerebrospinal fluid.

Host-pathogen interactions in bacterial meningitis.

Bacterial meningitis is a devastating disease occurring worldwide with up to half of the survivors left with permanent neurological sequelae. Due to intrinsic properties of the meningeal pathogens and the host responses they induce, infection can cause relatively specific lesions and clinical syndromes that result from interference with the function of the affected nervous system tissue. Pathogenesis is based on complex host-pathogen interactions, some of which are specific for certain bacteria, whereas others are shared among different pathogens. In this review, we summarize the recent progress made in understanding the molecular and cellular events involved in these interactions. We focus on selected major pathogens, Streptococcus pneumonia, S. agalactiae (Group B Streptococcus), Neisseria meningitidis, and Escherichia coli K1, and also include a neglected zoonotic pathogen, Streptococcus suis. These neuroinvasive pathogens represent common themes of host-pathogen interactions, such as colonization and invasion of mucosal barriers, survival in the blood stream, entry into the central nervous system by translocation of the blood-brain and blood-cerebrospinal fluid barrier, and induction of meningeal inflammation, affecting pia mater, the arachnoid and subarachnoid spaces.

The CcpA regulon of Streptococcus suis reveals novel insights into the regulation of the streptococcal central carbon metabolism by binding of CcpA to two distinct binding motifs.

Streptococcus suis (S. suis) is a neglected zoonotic streptococcus causing fatal diseases in humans and in pigs. The transcriptional regulator CcpA (catabolite control protein A) is involved in the metabolic adaptation to different carbohydrate sources and virulence of S. suis and other pathogenic streptococci. In this study, we determined the DNA binding characteristics of CcpA and identified the CcpA regulon during growth of S. suis. Electrophoretic mobility shift analyses showed promiscuous DNA binding of CcpA to cognate cre sites in vitro. In contrast, sequencing of immunoprecipitated chromatin revealed two specific consensus motifs, a pseudo-palindromic cre motif (WWGAAARCGYTTTCWW) and a novel cre2 motif (TTTTYHWDHHWWTTTY), within the regulatory elements of the genes directly controlled by CcpA. Via these elements CcpA regulates expression of genes involved in carbohydrate uptake and conversion, and in addition in important metabolic pathways of the central carbon metabolism, like glycolysis, mixed-acid fermentation, and the fragmentary TCA cycle. Furthermore, our analyses provide evidence that CcpA regulates the genes of the central carbon metabolism by binding either the pseudo-palindromic cre motif or the cre2 motif in a HPr(Ser)∼P independent conformation.