<p dir="ltr"><i>Stenotrophomonas maltophilia</i> is an emerging multidrug-resistant, Gram-negative opportunistic pathogen. It causes many hospital-acquired infections such as sepsis, endocarditis, meningitis, and catheter-related urinary tract infections. It also affects individuals with cystic fibrosis, exacerbating their lung condition. <i>S. maltophilia</i> often causes pathogenesis through the formation of biofilms. However, the molecular mechanisms <i>S. maltophilia</i> uses to carry out these pathogenic steps are unclear. The SMF-1 chaperone/usher pilus has been thought to mediate <i>S. maltophilia</i> attachment. To confirm this role, we created an isogenic deletion of the <i>smf-1</i> pilin gene and observed a defect in biofilm compared to wild type. We also discovered 2 additional chaperone/usher pilus operons, mutation of which also caused attenuation in biofilm levels. Analysis of <i>S. maltophilia</i> clinical strains and <i>S. maltophili</i><i>a</i> complete genomes listed in NCBI showed that these three pili are prevalent and highly conserved, suggesting a vital role in infection. Intriguingly, through TEM studies, we found that the mutation of one pilus is not phenotypically compensated by another. Infection of <i>Galleria mellonella</i> larvae revealed increased virulence of the pilus mutants. Additionally, we also demonstrated a relationship between pilus and flagella contributing to the overall biofilm development of <i>S. maltophilia</i>. Understanding their activity may help identify therapeutic targets for this pathogen.</p>

Yes / Recent studies portend a rising global spread and adaptation of human- or healthcare- associated pathogens. Here, we analyse an international collection of the emerging, multi-drug-resistant, opportunistic pathogen Stenotrophomonas maltophilia from 22 countries to infer population structure and clonality at a global level. We show that the S. maltophilia complex is divided into 23 monophyletic lineages, most of which harbour strains of all degrees of human virulence. Lineage Sm6 comprises the highest rate of human-associated strains, linked to key virulence and resistance genes. Transmission analysis identifies potential outbreak events of genetically closely related strains isolated within days or weeks in the same hospitals.

Cystic fibrosis (CF) is a fatal, incurable genetic disease that affects over 30,000 people in the United States alone. People with this disease have a homozygous mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) which causes defects in chloride transport and leads to build up of mucus in the lungs and disruption of function in various organs. CF patients often suffer from chronic bacterial infections within the lungs, wherein the bacteria persist as a biofilm, leading to poor prognosis. Two of these pathogens, <i>Stenotrophomonas maltophilia</i> and <i>Pseudomonas aeruginosa</i>, are often found in the lungs of patients with CF and are an increasing medical concerns due to their intrinsic antimicrobial resistance. Both species can readily form biofilms on biotic and abiotic surfaces such as intravascular devices, glass, plastic, and host tissue. Biofilm formation starts with bacterial attachment to a surface and/or adjacent cells, initiating the acute infection stage. Chronic, long-term infection involves subsequent or concurrent altered genetic regulation, including a downregulation of virulence factors, resulting in the bacteria committing to a sessile lifestyle, markedly different from the planktonic one. Many of these genetic switches from an acute to chronic lifestyle are due to pressures from the host immune system and lead to permanently mutated strains, most likely an adaptive strategy to evade host immune responses. Biofilms are extremely problematic in a clinical setting because they lead to nosocomial infections and persist inside the host causing long-term chronic infections due to their heightened tolerance to almost all antibiotics. Understanding the genetic networks governing biofilm initiation and maintenance would greatly reduce consequences for CF and other biofilm-related infections and could lead to the development of treatments and cures for affected patients. This study showed that in<i> S. maltophilia</i>, isogenic deletion of phosphoglycerate mutase (<i>gpmA</i>) and two chaperone-usher pilin subunits, <i>S. maltophilia</i> fimbrae-1 (<i>smf-1</i>) and<i> cblA</i>, lead to defects in attachment on abiotic surfaces and cystic fibrosis derived bronchial epithelial cells (CFBE). Furthermore, Δ<i>smf-1</i> and Δ<i>cblA</i> showed defects in long-term biofilm formation, mimicking that of a chronic infection lifestyle, on abiotic surfaces and CFBE as well as stimulating less of an immune response through TNF-α production. This study also showed that in <i>P. aeruginosa</i>, the Type III secretion system (T3SS), an important virulence factor activated during the acute stage of infection, is downregulated when <i>polB</i>, a stress-induced alternate DNA polymerase, is overexpressed. This downregulation is due to post-transcriptional inhibition of the master regulatory protein, ExsA. Taken together, this project highlights important genes involved in the acute and chronic infection lifestyle and biofilm formation in <i>S. maltophilia</i> and genetic switches during the acute infection lifestyle in <i>P. aeruginosa</i>.

Pseudomonas aeruginosa et Stenotrophomonas maltophilia, pathogènes opportunistes majeurs, pourraient acquérir leur résistance aux antibiotiques dans l'environnement, sous la pression exercée par les métaux lourds par co-sélection de résistance. Nous avons tout d'abord évalué la distribution et l'abondance de ces espèces dans un large panel de sols d'origine géographique différente (France et Afrique) et évalué l'influence d'activités anthropiques susceptibles d'exposer les sols en éléments métalliques sur cette distribution. Alors que la présence de P. aeruginosa est sporadique et plutôt liée à un apport exogène, S. maltophilia est présente dans tous les sols étudiés, suggérant son endémicité. L'évaluation des résistances des souches isolées de ces sols a également montré des différences entre les deux espèces. Les souches environnementales de P. aeruginosa sont pour la plupart caractérisées par un phénotype sauvage alors que celles de S. maltophilia présentent une grande diversité de phénotypes en fonction des sites, parfois similaires à ceux de souches cliniques. Cette diversité peut être attribuée à l'adaptation aux conditions environnementales très différentes rencontrées mais il est difficile d'attribuer précisément aux métaux un rôle dans la co-sélection de ces résistances. L'étude menée sur la communauté bactérienne d'un sol contaminé a également permis de mettre en évidence une forte proportion de bactéries résistantes à différents antibiotiques représentée par des espèces qualifiées de pathogènes opportunistes ainsi que la présence du gène blaIMP, permettant la résistance à l'imipénème, utilisé en milieu clinique pour le traitement de clones multi-résistants.

No description available.

4 ABSTRACT (English) This thesis is based on three published research papers studying bacterial REP (repetitive extragenic palindrome) elements. REP elements are one of the best-characterized groups of bacterial DNA repeats, distributed mostly in gammaproteobacteria, including enterobacteria. They are present in noncoding parts of host genomes, usually occurring in hundreds of copies. REPs are typically aggregated in higher order repeats. In the Gram-negative model Escherichia coli, interactions of several proteins important for cell's physiology with REPs were described, indicating significant role for these elements for host cells. The first work (Nunvar et al. 2010) presents the discovery of a protein class, related to IS200/IS605 transposases. These proteins, termed RAYTs (REP-associated tyrosine transposases), contain characteristic motifs in their amino acid sequences, which are absent in canonical IS200/IS605 transposases. Another attribute of RAYTs is the arrangement of their encoding genes. These are single copy genes, always flanked at both termini by at least two REPs in inverted orientation. Based on the similarity between the REP-rayt-REP unit and insertion sequences of the IS200/IS605 family, between RAYTs and tyrosine transposases and between REPs and subterminal sequences of the IS200/IS605...

No description available.

Gram-negative infection has been linked to hospital water although few studies have examined whether water systems are reservoirs of nosocomial pathogens. This study investigated longitudinal recovery of the opportunistic pathogens Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Acinetobacter baumannii from water outlets of a haematology unit and evaluated Point-Of-Use Filtration (POU-F) as a control measure. In a two-year double cross-over trial, water samples and swabs were taken weekly from 39 showers/taps on the unit. Four study phases alternated between non-filtered (Phases 1 &amp; 3), and filtered outlets (Phases 2 &amp; 4) using Pall AquasafeTM 14-day filters. In Phases 1 &amp; 3; 99% of 1396 samples yielded bacterial growth, with colonies generally too numerous to count. Target species were isolated from 22% of water samples (P. aeruginosa 14%; S. maltophilia 10%) and 10% of swabs. P. aeruginosa was particularly associated with handwash stations and S. maltophilia with showers. A. baumannii was not isolated. With POU-F; 22% of 1242 samples yielded bacterial growth (mean CFU/100ml ,4.6). S. maltophilia was isolated only once from water but never from outlet swabs. PCR typing identified clusters of isolates colonizing different outlets over time but no clear association between water and patient isolates was identified. The incidence of Gram negative infections remained low throughout the study. Without POU-F, water from taps/showers represented a source of bacteria including the target species. POU-F substantially reduced the frequency and number of target species from every outlet, and merits further investigation as an intervention to protect immunocompromised patients from opportunistic pathogens.

Occasionally, crystallisation of proteins works in mysterious ways! One might obtain crystals of a protein of unknown identity in place of the protein for which crystallisation experiments were performed. If the investigator is not aware of such possibilities, valuable time and resources might be lost in attempting to determine the structure of such proteins. Instances of non-target protein getting crystallised may not come to light at all or may be realised only when attempts to determine the structure completely fail by conventional procedures after collecting and processing the diffraction data. Usually, it is not possible to reproduce the crystals of the same protein as their occurrence is serendipitous. Such rare instances of crystallisation are probably caused by fluctuating environmental or crystallisation conditions and are not reproducible. It could also be due to contaminating microbes, which is more likely when the experimentalist is not well experienced. Therefore, experimental phasing of the data collected on serendipitously obtained crystals could be a challenging task. With the rapid increase in the number of structures deposited in the protein data bank (PDB), molecular replacement has become the method of choice for structure determination in macromolecular X-ray crystallography. This is due to the fact that it is possible to select a suitable phasing model for most target proteins based on their sequence information. However, if the identity of the target protein itself is uncertain, all attempts of structure determination using phasing models selected on the basis of target protein sequence-dependent search would fail. Sequence-independent ab initio phasing techniques such as ARCIMBOLDO (Meindl et al., 2012), which has recently become available, could provide leads only if the non-target protein is an all-α-protein and the associated diffraction data extends to a resolution better than 2 Å. Even then, the success rate with this technique is low. Hence, it becomes important to employ a sequence-independent method of structure determination for such mysteriously obtained crystals. This thesis reports crystal structures of proteins which are serendipitously crystallised using a large-scale application of Molecular Replacement (MR) technique (referred in this thesis as MarathonMR). This thesis also presents an evaluation of molecular replacement strategies for structure determination. The thesis begins with an overview of crystallographic methods of structure determination with an emphasis on the method of molecular replacement (Chapter 1). The most prominent of the results obtained in the course of these investigations pertains to a crystal obtained during routine crystallisation of a viral protein mutant in the year 2011. The cell parameters were different from cell constants of crystals obtained with other known viral protein mutants crystallised earlier in the same laboratory. Unfortunately, this crystal could not be reproduced in the same form in subsequent crystallisation trials. All attempts to determine the structure through conventional molecular replacement techniques using a combination of domains from a nearly identical virus coat protein protomer as the phasing model had failed. The data was shelved as “not-solvable” in late 2011. However, the crystal had diffracted to 1.9 Å and had excellent merging statistics. Therefore, the data was retrieved recently and additional attempts were made to determine the structure through phasing techniques that have become available recently. Techniques such as AMPLE (Bibby et al., 2013) and Rosetta (DiMaio, 2013), which use large-scale homology models coupled with molecular replacement, did not lead to meaningful solutions. A couple of helices identified by ARCIMBOLDO (Meindl et al., 2012) were neither correct (retrospectively) nor sufficient to determine the entire structure. Given the excellent merging statistics of the crystal data, there was significant motivation to determine the structure, though it meant developing a fresh protocol. It was at this time that we came across the work of Stokes-Rees and Sliz (2010) in which they had demonstrated that it is possible to determine structure of proteins of unknown identity by employing almost every known protein structure as a potential phasing model. The work reported in the thesis is a result of an earlier project to examine the relationship between properties of phasing models and the quality of target protein model generated through MR by employing large scale molecular replacement runs. This project was initiated because of the realisation that the recent explosion in crystallographic structural studies has resulted in near complete exploration of the “fold-space” of proteins and PDB now has a representative structure for most plausible folds of proteins. Some folds are highly represented in the PDB. Hence, it is likely that there would be at least one homologue in the PDB which could be used as a phasing model to successfully determine the structure of a protein of unknown identity if the diffraction dataset is of excellent quality. Hence, the single dataset which had diffracted to 1.9 Å resolution was used to develop a MarathonMR procedure for structure determination. MarathonMR procedure takes sequence-independent approach to structure determination and employs large-scale molecular replacement calculations to identify the closest homologue (in structural terms initially). This protocol is described in Chapter 2 (Materials and methods) of the thesis. Through MarathonMR, structure of the dataset which had remained unsolved for 5 years was finally determined. Nearly complete sequence of the polypeptide could be deduced by inspecting the electron density map due to the high resolution and quality of the map. The protein was found to be a phosphate binding protein from a soil bacterium Stenotrophomonas maltophilia (SmPBP). The way in which the structure was determined and possible explanations for the mysterious source of this protein which had crystallised instead of the target protein is discussed in Chapter 3. Though MarathonMR procedure was developed to solve a single dataset, it was soon realised that the same procedure could be applied to other similar datasets, all of which had diffracted to reasonable resolutions with good merging statistics but had remained unsolved for unknown reasons. Among such datasets, one of the datasets which was collected in 2007 and had diffracted to 2.3 Å resolution had cell parameters very close to that of SmPBP. Hence, a poly-alanine model of the structure of SmPBP, which was determined by then, was used as the phasing model to run molecular replacement and the structure was readily solved. It was surprising to note that SmPBP had crystallised serendipitously not once but twice, once in 2011 resulting in crystals that diffracted to 1.9 Å resolution and earlier in 2007 in crystals that diffracted to 2.3 Å resolution independently by two different investigators in the same laboratory. Both the structures are nearly identical and a comparison of these structures is presented in Chapter 4. Structure of SmPBP determined at 2.3 Å resolution by MarathonMR also corresponds to the dataset that had remained unsolved for the longest period of time (9 years). This success of structure determination after the lapse of such a long period emphasises the importance of carefully preserving X-ray diffraction data irrespective of its immediate outcome. In Chapter 5 of the thesis, another instance of non-target protein crystallisation, the structure of which was determined using the MarathonMR procedure is described. The crystal was obtained while carrying out crystallisation of mutants of a survival protein (SurE) expressed in Salmonella typhimurium when the bacterium is subjected to environmental or internal stresses. The original investigator had used the structure of SurE as the phasing model to determine structure of the mutant crystals and obtained a model with R and Rfree of 35% and 40%, respectively. However, the model did not refine further to lower R-factors suggesting that the solution obtained may not be correct. MarathonMR indicated that the fold of the crystallised protein could be similar to that of glycerol dehydrogenase. As SurE shares some fold similarity with one of the domains of GlyDH, the original investigator might have been able to achieve a limited success with R/Rfree factors of 35% and 40%, respectively. As the merging statistics for this diffraction data set was poor, the diffraction images were reprocessed in XDS program on Xia2 automated spot processing pipeline. The data statistics indicated merohedral twinning (14%). However, using appropriate parameters, it was possible to refine the structure obtained by MarathonMR to acceptable R/Rfree using the Refmac program. Four protomers were present in the crystal asymmetric unit (ASU). Non-crytsallographic symmetry averaging of electron density over these four molecules further improved the electron density. As the data was limited to 2.7 Å resolution, it was not possible to deduce the identity of every residue of the protein unambiguously based solely on the resulting electron density map. With the identity of the amino acids that could be deduced with certainty, it was clear that the protein belongs to glycerol dehydrogenase from a species of Enterobacteriacea family. Though a similar structure of glycerol dehydrogenase has been reported from Serratia, there are clear differences in many unambiguously determined residues which suggest that the protein is not from Serriatia. The protein has been named EnteroGlyDH as the source of the protein is likely to be from a species of Enterobacteriacea family. The structure of the protein, its biochemical implications and possible reasons for the serendipitous crystallisation of a non-target are discussed. Chapter 6 discusses the structure determination of an inorganic pyrophosphatase and catalytic domain of Succinyl transferase, the crystals of which had diffracted to 2.3 Å and 3.1 Å, respectively, but had remained unsolved. Neither of the datasets corresponds to the intended target proteins. The dataset corresponding to the protein whose structure was determined as that of an inorganic pyrophosphatase was provided by a colleague from a different laboratory in the Indian Institute of Science. It is interesting to note that the investigator had carried this dataset to one of the CCP4 workshops and had tried to determine the structure with the help of experts in the workshop. The attempts to determine its structure had however failed for reasons that are obvious now. The original investigator was unfortunately making efforts with an erroneous assumption on the identity of the target protein. As these enzymes are well studied, their structures and functions are briefly discussed. It is already well established that molecular replacement is being used with increasing frequency as the phasing technique when compared to other experimental phasing techniques. With the ever growing number of structures in the PDB, high population of certain folds and a near-plateau attained in the identification and growth of new folds, it is reasonable to expect that molecular replacement will be used even more frequently in the years to come. Therefore, for carrying out molecular replacement for a given diffraction dataset of a target protein, it is very likely that several homologous structures would be available in the PDB that could be used as potential phasing models. Hence, it becomes important to understand the influence of phasing model on the quality and accuracy of model generated through MR to achieve the best structure solution. To understand this relationship between phasing model and model obtained by MR protocol, re-determination of already known structures deposited in the PDB starting with their respective structure factors and various phasing models was initiated. Structures belonging to TIM beta/alpha-barrel (SCOPe ID: c.1) and Lysozyme-like (SCOPe ID: d.2) folds were chosen as targets. The structure of each target was re-determined serially starting with poly-alanine models of all available unique homologues as phasing models. Due to the multi-dimensional nature of this study, the results obtained were represented in a graphical form with nodes and edges. Detailed methodology of the work carried out and the data representation model are discussed in the Chapter 2 (Materials and methods). It was found that after a certain sequence identity cut-off, sequence identity between phasing model and target seems to have little influence on the quality and accuracy of the model generated through MR. Instead, other qualities of the phasing model such as Rfree and RSCC influence the quality of MR models. These results are discussed in Chapter 7. Learning from the work reported in this thesis are discussed in concluding chapter. The possible logical and programmatic upgrades to MarathonMR protocol and future path in which the relationship between phasing models and models generated through MR can be studied are discussed in Chapter 8 (Conclusion and future prospects).

No description available.