Long-term Stationary Phase Behavior of Streptococcus pyogenes Biofilms Department of Microbiology and Immunology Streptococcus pyogenes is the etiological agent of many human diseases ranging from mild superficial skin infections and pharyngitis to life-threatening necrotizing fasciitis. There can be several complications as a result of S. pyogenes infection including post-streptococcal glomerulonephritis and rheumatic fever, which leads to rheumatic heart disease. Despite the significant virulence associated with the pathogen, the bacteria can also persist asymptomatically in human host carriers. S. pyogenes is characterized by significant strain-to-strain variation with many single nucleotide polymorphisms and differences in genetic content of up to 33% of the genome. Active infection is associated with the rapid growth of the pathogen, whereas survival or carriage is associated with slow growth. Our laboratory has demonstrated that during survival in long-term stationary phase cultures and in eukaryotic cells, S. pyogenes diversifies into a mixed population. Isolates from this population show diversification in their proteome, in metabolism, and in virulence factor transcription patterns. These are stable, heritable changes with unique mutations in global gene regulators in some isolates, suggesting that an accumulation of genetic mutations leads to diversification. There are two proposed modes of survival in the human host; by taking residence intracellularly in host cells and as biofilms. Previous studies showed that isolates surviving within eukaryotic cells acquire heritable changes in metabolism and virulence factor expression. Biofilms are highly organized structures formed by many bacteria, which provide resiliency to harsh environmental conditions. It has been demonstrated that S. pyogenes form biofilms in vivo and in vitro, and up to 90% of clinical isolates can form biofilms. Considering the resiliency of biofilms, and the organized roles played by individual cells in biofilms, we hypothesized that biofilms may provide S. pyogenes with a niche for persistence and diversification. Despite the capacity for survival of planktonic cells, we have found that viable cells could not be isolated from static biofilms after 10 days. No metabolic variants were found among biofilm isolates prior to loss of biofilm viability. Biofilm structure was examined using confocal microscopy to image cells after LiveDead® staining. These experiments revealed that the biofilms lost viability rapidly, and also appeared to disperse. Dispersion of 2-day old biofilms could be induced with culture supernatants collected from 7-day old planktonic cells. Overall, the results of these studies suggest that secreted factors from late stationary phase cultures induce biofilm dispersion and biofilms do not serve as a niche for long-term survival and diversification of S. pyogenes. Therefore, S. pyogenes biofilms may be more critical for initial colonization of the oropharynx. These studies may provide a valuable insight to the role of biofilms in S. pyogenes infections. / Microbiology and Immunology

Streptococcus pyogenes is a human-specific pathogen that can cause a wide variety of diseases. These diseases range from the relatively mild pharyngitis and impetigo to invasive diseases such as necrotizing fasciitis to post-streptococcal sequelae such as rheumatic heart disease. The bacteria are frequently carried asymptomatically and may cause recurrent disease. Corresponding with their etiologic variation amongst diseases, clinical isolates demonstrate diverse virulence factor expression and random genetic mutations. In these studies, we examine the role of intracellular residence during survival as a niche for the diversification of S. pyogenes. Survival was previously studied using two in vitro systems: long-term stationary phase survival in culture and survival within epithelial cells in the presence of extracellular antibiotics. The surviving populations diversified, giving rise to stable strains with alternate colony morphologies, distinct proteomes, and altered metabolic properties. Further analysis in these studies showed that alterations in colony morphology were not solely observed during survival, but could also be induced in models mimicking acute infection. However, diversification in certain metabolic pathways occurred only during survival, and this metabolic diversification was observed at the transcriptional level. Further, one of three clinical isolates from patients with recurrent pharyngitis was altered in its metabolic profile, suggesting metabolic diversification may be occurring in vivo. The survivor strains had varied transcriptional changes in the genes encoding the virulence factors emm, slo, and speB. All of the stationary phase-derived survivor strains and two intracellular survival-derived strains had attenuated virulence in zebrafish. Most of the attenuated strains disseminated to the spleen and were cleared within three days. A whole blood killing assay showed a strong correlation between bacterial killing and emm expression. While the diversification appeared random, these strains retained their multilocus sequence type (MLST). These results suggest S. pyogenes strains with the same MLST, but diverse virulence properties, may arise during survival in the host. / Microbiology and Immunology

The upper respiratory tract (URT), including the nasal and oral cavities, is a reservoir for pathogenic and commensal microbial species, collectively known as the microbiota. Microbial colonization of the URT occurs right after birth, and URT microbial composition has been linked to development of respiratory infections, allergy, and asthma, though few direct mechanisms have been uncovered. Thus, I set out to establish animal models for characterizing the URT microbiota, and its role in infections. I found that nasal washes, a predominant method for measuring URT bacterial colonization, were insufficient for completely extracting the URT microbiota. The age and source of mice greatly affected the composition of the microbiota, which could be transferred to germ-free mice via cohousing. I also established that mice colonized with the Altered Schaedler’s Flora in the gut microbiota have no cultivable URT microbiota. To test the function of the URT microbiota, I colonized mice with Streptococcus pneumoniae, the leading cause of bacterial pneumonia worldwide. I show that the presence of a nasal microbiota increases permissiveness to pneumococcal infection in murine models. Addition of a single URT isolate, Actinomyces naeslundii, increased pneumococcal adherence to human respiratory epithelial cells in vitro and increased pneumococcal dissemination in vivo in a sialidase-dependent manner. The microbiota affects expression of several host genes throughout the respiratory tract involved in pneumococcal pathogenesis. Together, this work establishes new models for assessing the URT microbiota, and highlights the contribution of the URT microbiota to pneumococcal pathogenesis and identifies druggable targets to prevent and treat infections. / Dissertation / Doctor of Philosophy (PhD) / Bacteria living in the gut have been shown to benefit our health, but the role of bacteria living in our respiratory tract is relatively unknown. I describe the methods for characterizing the bacteria in the nose of a mouse as a model of the human nose. I found that pockets of the mouse nose are colonized by different bacteria. I also characterized a mouse model that had bacteria in the gut without nasal bacteria. I used this mouse model to understand infections with Streptococcus pneumoniae, the worldwide leading cause of bacterial pneumonia. The mice without nasal bacteria were protected from infections, which was due to a nasal bacteria helping S. pneumoniae escape from the nasal tissue. This work established new models for understanding how bacteria affect respiratory health, and identified new targets for protecting against infections.

Streptococcus pneumoniae (pneumococcus) is a Gram positive opportunistic bacterium that is a primary cause of pneumonia in young children and immunocompromised individuals. This microorganism colonizes the nasopharynx of all age groups and health levels and is easily transmitted through respiratory droplets or aerosols. While capsule-based vaccines are available, these are becoming more obsolete as S. pneumoniae strains are undergoing serotype replacement, thus evading detection, and antibiotic resistant strains are increasing in prevalence. This study has taken a different approach by identifying immunogenic proteins that elicit antibodies which block attachment of bacteria to host epithelial cells. Proteins with the most potential were assessed by analyzing the reactivities of human sera with varying degrees of colonization blockage. Proteins which reacted with the primary protective antibody in mucosal sites, IgA, were prioritized. Identification of those proteins through mass spectrometry will be crucial in creating more effective, long-term protection against S. pneumoniae infections.

Streptococcus pneumoniae (pneumococcus) is a commensal gram-positive colonizer of the human nasopharynx capable of causing diseases including otitis media, pneumonia, bacteremia, and meningitis. Although it is often a harmless colonizer, there is a high rate of mortality and morbidity among the immunocompromised, elderly, and young children. While these infections can often be treated with antibiotics, resistance to numerous antibiotics is increasing. Antibiotic resistance is a well-studied dilemma; however, little information is known of how bacteria take up certain antibiotics. Because most antibiotics cannot diffuse freely across the bacterial cell wall, we hypothesize that metabolite transport proteins participate in the uptake of certain classes of antibiotics.

No / Streptococcus agalactiae is a major human and animal pathogen, most notable as a cause of life-threatening disease in neonates. S. agalactiae is also called the Group B Streptococcus in reference to the diagnostically significant Lancefield Group B typing antigen. Although the structure of this complex carbohydrate antigen has been solved, little is known of its biosynthesis beyond the identification of a relevant locus in sequenced S. agalactiae genomes. Analysis of the sugar linkages present in the Group B carbohydrate (GBC) structure has allowed us to deduce the minimum enzymology required to complete its biosynthesis. Most of the enzymes required to complete this biosynthesis can be identified within the putative biosynthetic locus. Surprisingly, however, three crucial N-acetylglucosamine transferases and enzymes required for activated precursor synthesis are not apparently located in this locus. A model for GBC biosynthesis wherein the complete polymer is assembled at the cytoplasmic face of the plasma membrane before translocation to the cell surface is proposed. These analyses also suggest that GBC is the major teichoic acid-like polymer in the cell wall of S. agalactiae, whereas lipoteichoic acid is the dominant poly(glycerophosphate) antigen. Genomic analysis has allowed us to predict the pathway leading to the biosynthesis of GBC of S. agalactiae.

No / Streptococcus equi is the causative agent of strangles, a prevalent and highly contagious disease of horses. Despite the animal suffering and economic burden associated with strangles, little is known about the molecular basis of S. equi virulence. Here we have investigated the contributions of a specific lipoprotein and the general lipoprotein processing pathway to the abilities of S. equi to colonize equine epithelial tissues in vitro and to cause disease in both a mouse model and the natural host in vivo. Colonization of air interface organ cultures after they were inoculated with a mutant strain deficient in the maturase lipoprotein ( prtM138-213, with a deletion of nucleotides 138 to 213) was significantly less than that for cultures infected with wild-type S. equi strain 4047 or a mutant strain that was unable to lipidate preprolipoproteins ( lgt190-685). Moreover, mucus production was significantly greater in both wild-type-infected and lgt190-685-infected organ cultures. Both mutants were significantly attenuated compared with the wild-type strain in a mouse model of strangles, although 2 of 30 mice infected with the lgt190-685 mutant did still exhibit signs of disease. In contrast, only the prtM138-213 mutant was significantly attenuated in a pony infection study, with 0 of 5 infected ponies exhibiting pathological signs of strangles compared with 4 of 4 infected with the wild-type and 3 of 5 infected with the lgt190-685 mutant. We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive pathogen in its natural host. These data suggest that the PrtM lipoprotein is a potential vaccine candidate, and further investigation of its activity and its substrate(s) are warranted.

No / Streptococcus mutans was shown to produce two extracellular proteases capable of degrading both gelatin and collagen-like substrates. These enzymes have molecular masses of 52 and 50 kDa when analysed by SDS-PAGE. Both enzymes were inhibited by EDTA, but not by a range of other inhibitors with different specificities, indicating that they are metalloproteases. The activity of EDTA-inactivated enzymes could be restored by the addition of manganese and zinc. The identical inhibition and restoration profiles of the two enzymes suggest that one of the proteases may be a degradation product of the other.

No / Isogenic mutants of Streptococcus mutans LT11, deficient in the production of the wall-associated protein antigens A and B, were generated by recombinant DNA technology. The hydrophobicity, adherence, and aggregation of the mutants were compared with those of the parent strain. These studies indicated that hydrophobicity, adherence, and saliva- or sucrose-induced aggregation were unaltered in the A- mutant but that hydrophobicity and adherence to saliva-coated hydroxylapatite were greatly reduced in the B- mutant whilst sucrose-dependent adherence and aggregation were increased. To determine whether these changes correlated with changes in the mutated gene product alone, the levels of a number of cell wall antigens were determined in each of the mutants. The loss of antigen A resulted in significantly reduced levels of wall-associated lipoteichoic acid, and loss of antigen B resulted in reductions in both antigen A and lipoteichoic acid. Data presented here thus suggest that changes in the expression of one wall antigen can have a dramatic effect on the levels of others.

Streptococcus pneumoniae est le pathogène bactérien le plus important des voies respiratoires chez les adultes et les enfants causant la pneumonie, la bronchite et l’otite de l’oreille moyenne. Cette bactérie est responsable d’une morbidité et d’une mortalité importante, entre autres, chez les jeunes enfants. La prévalence générale des souches de pneumocoques résistants et multirésistants est en hausse dans le monde compliquant la thérapie antimicrobienne vis-à-vis cette bactérie. Face à cette problématique, nous avons voulu caractériser les mécanismes de résistance à la ciprofloxacine (CIP), la tétracycline (TC) et la tigécycline (TGC) chez des mutants sélectionnés en laboratoire et des souches cliniques afin de potentiellement découvrir de nouvelles cibles diagnostiques de la résistance vis-à-vis ces molécules. L’approche génomique utilisée dans cette thèse (séquençage de génome et transformation) a permis de faire la caractérisation génotypique et phénotypique des mutants résistants aux trois antibiotiques utilisés dans l’étude. Cette approche, en plus de préciser le rôle des mutations dans les gènes parC et gyrA dans la résistance à la CIP, a permis de déterminer que le pneumocoque peut mettre en place des mécanismes de résistance secondaires le rendant résistant à la CIP et à la TC. En effet, l’acquisition d’une mutation dans le gène spr1902 protège la bactérie contre les dérivés réactifs à l’oxygène (ROS) induit par la CIP. De plus, la surexpression de PatA/PatB ainsi que la présence de mutations dans l’opéron du transporteur PatA/PatB induit de faibles niveaux de résistance à la CIP et à la TC, en absence de tetM et tetO. Un lien a également été établi entre la résistance à la TC et la surexpression de gènes de la voie de biosynthèse de la thiamine chez des souches de S. pneumoniae non-sensibles à la TC. Finalement, les mécanismes de résistance à la TGC ont été décrit, pour la première fois, chez le pneumocoque (mutations dans la protéine ribosomale S3 (rpsC ; spr0195), S10 (rpsJ; spr0187), l’ARNr 16S et une méthyltransférase de l’ARNr 16S hypothétique (spr1784). Ceuxi-ci causent une résistance croisée aux TCs de première et deuxième génération. Dans cette thèse, nous avons mis en lumière de nouveaux marqueurs de la résistance aux antibiotiques chez S. pneumoniae. / Streptococcus pneumoniae is a Gram-positive pathogen responsible for pneumonia, bronchitis and otitis media leading to considerable morbidity and mortality among children and adults. The prevalence of resistant and multi-resistant strains increases worldwide impairing antimicrobial treatments toward this bacterium. We characterised resistance to ciprofloxacin (CIP), tetracycline (TC) and tigecycline (TGC) in laboratory-derived resistant mutants and unsusceptible clinical isolates to further our comprehension of resistance mechanisms and potentially uncover new therapeutic and diagnostic targets toward these drugs. The genomic approaches used in this thesis (genome sequencing and DNA transformation) allowed the phenotypic and genotypic characterisation of mutants resistant to three antibiotics. By this approach, the role of parC and gyrA mutations in CIP resistance was confirmed and even extended and it was also possible to determine that S. pneumoniae may select secondary mechanisms of resistance to CIP and TC besides target-site mutations. Acquisition of a mutation in spr1902 is shown to protect the bacteria against oxygen-reactive species induced by CIP. Furthermore, overexpression of the ABC transporter PatA/PatB and mutations in the coding region of this transporter confer low-level resistance to CIP and TC. A link was also established between TC resistance and overexpression of genes involved in the thiamine biosynthesis and salvage pathway in S. pneumoniae TC non-susceptible isolates. Finally, for the first time, the mechanisms of resistance to TGC in S. pneumoniae were described (mutations in ribosomal protein S3 (rpsC; spr0195), S10 (rpsJ: spr0187), 16S ribosomal RNA (rRNA) and a putative 16S rRNA methyltransferase). These confer cross-resistance to first and second generation TCs. This work highlights new markers of antibiotic resistance in S. pneumoniae.