Yersinia pestis, the causative agent of plague, specializes in causing dense bacteremia following intradermal deposition of a small number of bacteria by the bite of an infected flea. This robust invasiveness requires the ability to evade containment by the innate immune system. Of the various mechanisms employed by Y. pestis to subvert the innate immune response and to proliferate rapidly in mammalian tissue, only a few are well-characterized. Here, I present two complementary genetic analyses of Y. pestis adaptations to the mammalian environment. In the first, genome-wide fitness profiling for Y. pestis by Tn-seq demonstrates that the bacterium has adapted to overcome limitation of diverse nutrients during mammalian infection. In the second, a series of combinatorial targeted mutations disentangles apparent functional redundancy among the effectors of the Y. pestis type III secretion system, and we report that YpkA, YopT, and YopJ contribute to virulence in mice. We have also begun to investigate a novel relationship between Y. pestis and mammalian platelets, a highly abundant cell type in plasma. I present evidence that Y. pestis has evolved specific mechanisms to interfere with platelet activation, likely in order to evade immune responses and promote maintenance of bacteremia by undermining platelet thrombotic and innate immune functions. The principles guiding this work – systematic genetic analysis of complex systems, coupled with rational modification of in vitro assays to more closely mimic the in vivo environment – are a generalizable approach for increasing the efficiency of discovering new virulence determinants in bacterial pathogens.

La résistance bactérienne aux antibiotiques est un problème de santé publique majeur, touchant plus particulièrement les enfants dans les pays en développement (PED).Nous avons effectué une revue systématique de la littérature pour quantifier le niveau de résistance aux antibiotiques chez les enfants âgés de moins de 2 ans dans les PED. De manière générale, les données sur la résistance aux antibiotiques dans la population étudiées sont rares. Selon les publications identifiées, Staphylococcus aureus, Escherichia coli, et Klebsiella spp. apparaissent comme les causes les plus fréquentes d’infections néonatales sévères. Chez les enfants âgés de 1 à 24 mois, Streptococcus pneumoniae et Salmonella spp. apparaissent comme les causes les plus fréquentes d’infections bactériennes invasives.Dans une seconde revue systématique, nous avons examiné les méthodologies actuelles utilisées pour mesurer la consommation d’antibiotiques dans les PED.Nos résultats montrent qu’aucunes des méthodologiesne permet, à elle seule, de répondre aux besoins de ces pays en terme de données.Nous avons conduit une enquête en population à Madagascar et au Sénégal afin d’examiner les modalités de consommation d’antibiotiques chez des enfants de moins de 2 ans. Dans les 2 pays, la plupart des antibiotiques étaient achetés en pharmacie sur présentation d’une ordonnance. Une proportion élevée des antibiotiques était utilisée pour le traitement d’infections probablement d’origine virale. Des facteurs tels que la disponibilité de centres de santé, de pharmacies, l’existence de programmes de remboursement ou encore la formation du personnel pourraient influencer la fréquence de consommations d’antibiotiques au niveau national.Les résultats issus de ces travaux de recherche ajoutent des données essentielles à la littérature existante et mettent en évidence des leçons importantes pour la lutte contre la résistance aux antibiotiques dans les PEDs. / Antimicrobial resistance is a growing threat across the world and is likely to disproportionately affect children in low-income countries (LICs).To estimate the burden of antibiotic resistance in the community among children under two in LICs we undertook a review of published literature. Common isolates in neonatal sepsis cases included Staphylococcus aureus, Escherichia coli, and Klebsiella. Among children 1 mo. to 2 yrs., Streptococcus pneumonia and Salmonella were most often reported. Information on antibiotic resistance was sparse and often relied on few isolates.We reviewed methods to measure antibiotic consumption in LICs from published literature and showed that current techniques used in isolation are insufficient to respond to all the data needs in LICs. Integrating study techniques and starting with community surveys may respond more adequately to this issue in LICs and lead to more actionable results.To investigate patterns of antibiotic consumption and related factors among children under two in Madagascar and Senegal we undertook community surveys in two sites in Madagasgar (Antananarvo and Moramanga) and one site in Senegal. Results showed relatively high levels of antibiotic use among children. The majority of antibiotics were purchased in pharmacies with a prescription in both countries. Data suggest a high proportion of use for likely viral infections. Local contexts including the availability of health care facilities, availability of pharmacies, national payment schemes, and provider training seemed to play a role in country usage rates.Results from this work add essential data to the literature where relatively little data exists and reveal important lessons about studying and combating antibiotic resistance in LICs.

The primary objective of this thesis is to differentiate between the risk factors for pre-and post-monsoon cholera epidemics in rural Bangladesh by analyzing the complex interaction between select environmental, cultural/behavioral, and socioeconomic variables over space and time. In rural Bangladesh, cholera epidemics correspond with the annual monsoon: the first, and smallest, occurs between March and June, while the larger cholera peak occurs between September and December. The differences between the spatial and temporal patterns of seasonal cholera are analyzed, and the risk factors are calculated for pre-and post-monsoon cholera epidemics. The theoretical approach that underlies this medical geographical study is disease ecology, which espouses that risk of disease is caused by an interaction between people and their environment. This thesis is structured around a holistic understanding that human-environment interactions are inseparable. In Bangladesh, the monsoon season typically starts between May and June. The 1992 and 1993 cholera peaks occurred just before the monsoon in April and March respectively, while the 1994 cholera peak occurred between April and June. In 1992 and 1993 cholera incidence increased in the post-monsoon period, and peaked in October. The 1994 post-monsoon cholera peak occurred in November. There is a regular temporal pattern to cholera, as the peaks followed a seasonal pattern with the smaller epidemic occurring in the pre-monsoon period and the larger epidemic occurring in the post-monsoon period. This study shows that there are different risks associated with pre-monsoon cholera epidemics and post-monsoon cholera epidemics. The two main risk factors associated with cholera incidence pre-monsoon were bari population (i.e., crowding) and a house located within the flood controlled area. These two variables were even more strongly associated with post-monsoon cholera incidence to a greater degree, along with a number of other variables including water use, sanitation practices, and socioeconomic status.

Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis, has been a global health problem for years. The emergence of drug resistance in this organism generates the necessity of exploring novel targets and developing new drugs. Topoisomerases are enzymes found in all kingdoms of life responsible for overcoming the topological barriers encountered during essential cellular processes. The genomes of mycobacteria encode only one type IA topoisomerase (MtopI), which has been validated as a novel TB drug target. The goal of this study is to obtain new information on the mechanism and resistance of endogenous and synthetic inhibitors of MtopI. Rv1495 is a M. tuberculosis toxin that belongs to the MazEF family (MazE is the antitoxin and MazF is the toxin), with endoribonuclease activity. Rv1495 (MazF homolog in M. tuberculosis) toxin has been shown to interact directly with the C-terminal domain of MtopI for mutual inhibition. In this study the interaction of Rv1495 with the positively charged C-terminal tail in Mtop I is reported. This new information is useful for rational design and discovery of antibiotics for mycobacteria. Ethacridine, an FDA approved drug has shown activity against MtopI. In this project we studied the mechanisms of resistance associated with this drug as well the use of Ethacridine in combination with Moxifloxacin, to potentiate the bactericidal effect of this current second line drug for TB treatment. Results from sequencing of the genomic DNA isolated from the resistant mutants suggested the involvement of the Holliday-junction Ruv resolvase. Further studies showed that co-treatment with Ethacridine can enhance the moxifloxacin-mediated killing of M. smegmatis. FP-11g, a novel fluoroquinophenoxazine inhibitor of bacterial topoisomerase I, has shown promising activity against M, tuberculosis. We explored the bactericidal activity and resistance mechanisms associated to FP-11g using M. smegmatis as model organism. Additionally, the inhibitory effect of FP-11g was also evaluated on M. abscessus. FP-11g at concentration 4X MIC showed complete bactericidal activity against M. smegmatis after 24 hours. Inhibitory activity against M. abscessus was also observed. Results from sequencing of the genomic DNA isolated from the M. smegmatis resistant mutants revealed mutations in genes associated with general drug resistance.

The breadth of genetic diversity in the mammalian immune response stands out amongst the ubiquity of variation seen in the genome, evidence that microbial infections have been a major driver of evolution. As technology has facilitated an understanding of the etiology of immunological diversity, so too has it enabled the assessment of its varied functions. Functional genomics, with its ability to assess both cause and effect, has revolutionized our understanding of fundamental biological phenomena and recalibrated our hypotheses. We build upon the model of host immunity established by rare genetic variants that are causative of immunodeficiencies, but that incompletely consider the complexities of the genome. To expand our understanding, we performed a series of forward genetic screens to identify regulators of distinct functions of the innate immune system. Our studies discovered genes with novel functions in antigen presentation and immunoregulation, including several involved in central metabolism. Studies in macrophages and dendritic cells identified mitochondrial respiration as a positive regulator of the interferon-gamma response, and cells incapable of respiration failed to activate T cells. Notably, human mutations in several of these genes are responsible for immune dysfunction. In summary, this work uses new methods in genetic engineering to systematically assess the regulation of innate immunity. Our results suggest that variation in these regulatory pathways is likely to alter immunity in states of health and disease. Thus, our work validates a new approach to identify candidate genes relevant to immune dysfunction.

The diploma thesis deals with droplet infections and finds out what knowledge, attitudes and behavior pupils of the lower secondary school have towards them. The theoretical part focuses on educational documents in the Czech Republic, including the occurrence of topics about droplet infections in RVP ZV, didactic game as a teaching method and a detail description of selected viral and bacterial droplet infections. In the practical part, the diploma thesis finds out what knowledge, attitudes and behavior students have about droplet infections. Subsequently, a didactic game is performed and the effectiveness of the didactic game is evaluated by comparing the pre-test and the post-test on the basis of changes in the knowledge, attitudes and declared behavior of the interviewed pupils. The results showed that due to the didactic game there was an improvement in the students knowledge and a desired change of attitudes. The declared behavior of students remained unchanged, as the entry level of students in this area was already at a high level.

For several decades amphibian populations have been declining. Historically, the bacterium A. hydrophila (Ah) was hypothesized to be the causal factor in amphibian disease and population declines. However, with the discovery of a chytrid fungus, Batrachochytrium dendrobatidis (Bd) in 1998, which was identified on the skin of amphibians during documented mortality events, Ah research became of minor interest as focus shifted to Bd. Recent studies into the immunocompromising abilities of Bd, however, have opened new questions about its relationship with Ah and their combined effects on a host. In this study, I explore the relationship between infection with these two pathogens, Bd and Ah, in two amphibian species from distinct regions of the United States. I developed a novel qPCR assay to measure the microbial load of Ah on the skin of two anuran species, Lithobates yavapaiensis (N=232) and Pseudacris ornata (N=169), which have confirmed Bd infections. I use a logistic regression model to identify whether significant relationships exist between these two pathogens, disease, and death. I find that even amongst the most severely infected frogs, Ah is not detectable on the skin and only appears post-mortem. I therefore conclude that Ah is an opportunistic bacterial pathogen, scavenging on anurans only after mortality events. This research is the first known study to quantitatively assess Ah in amphibians in conjunction with Bd. While there is no causal relationship between these pathogens, future work will examine potential Ah infections in other organs to more fully understand the relationship between Bd and Ah.

37.3 million Americans (11.2% of the US population) currently have Type 2 diabetes mellitus (T2DM) with over 1.5 million new cases being diagnosed each year. The multifactorial etiology of the patient having neuropathy, overweight/obesity, foot deformities, ischemia, and infection leads to a condition called diabetic foot ulcer (DFU). One in six patients with a DFU will require amputation with infected DFUs have a 155-fold increased risk of amputation. Staphylococcus aureus is the most common bacteria isolated from severe DFU infections that require amputation. Interestingly, diabetics are more heavily colonized with S. aureus compared to non-diabetics suggesting a unique advantageous adaptation to diabetes. The specifics of the underlying molecular mechanisms and triggers by which S. aureus adapts and thrives in the T2DM patient that increase its pathogenicity and colonization compared to non-diabetics with skin ulcer infections are not fully elucidated. Thus, our studies aimed to identify the key virulence components in the pathogenesis of S. aureus infected DFUs and using that information to develop therapeutics aimed at disrupting these components to increase the success rate of conservative treatment and prevent non-traumatic lower extremity amputations in T2DM patients. Our studies found that several different elevated sugars in T2DM patients can trigger virulence factor production in S. aureus. We also found by comparing several different clinical DFU S. aureus isolates that there are clear differences in the ability of each isolate to cause necrotic infections. And lastly, we identified a possible therapeutic, the amino acid L-arginine, that can help prevent/treat S. aureus infections in the Tallyho diabetic mouse model. In conclusion, we have increased the understanding of the pathogenesis of S. aureus infected DFU and have proposed a possible therapeutic to add to the conservative treatment regimen.

[ES] La cristalografía de rayos X es una potente técnica para la resolución de la estructura atómica de macromoléculas. La información generada, tiene gran impacto sobre diferentes campos relacionados con la investigación básica y aplicada, como son la biomedicina y diseño de fármacos, al igual que en el desarrollo de aplicaciones nanotecnológicas y biotecnológicas. Esta Tesis se centra en determinadas problemáticas actuales y en las proteínas involucradas en las mismas (TryR, eEF1A2 y CBDP35), siendo éstas sujeto de desarrollo biotecnológico en los campos de la biomedicina, farmacia y de la industria alimentaria, en el que la cristalografía de rayos X juega un papel crucial para dilucidar sus estructuras atómicas y funciones. En consideración a la biomedicina y diseño de fármacos, hemos resuelto la estructura de la Tripanotión reductasa (TryR) de Leishmania infantum en complejo con potentes inhibidores de su actividad oxidorreductasa, con potencial de desarrollo como fármacos. Así, se ha caracterizado la unión y mecanismo de acción de éstos inhibidores. TryR es una reconocida diana farmacológica para el tratamiento de la enfermedad de Chagas, la Tripanosomiasis Humana Africana y la leishmaniosis, ya que desempeña un papel crucial y esencial en el metabolismo redox de los parásitos de la familia Trypanosomatidae. Además, se han analizado los parámetros de cristalización y difracción de novedosos inhibidores de la dimerización de TryR, cuyo diseño racional se basa en la unión a la interfaz de dimerización de la misma. La oncoproteína eEF1A2, involucrada en múltiples funciones celulares y sujeto de numerosas modificaciones post-traduccionales, se une al fármaco anticancerígeno plitidepsina. La cristalografía de rayos X, combinada con experimentos de espectrometría de masas, se han utilizado como herramientas para identificar nuevas modificaciones post-traduccionales y características estructurales en eEF1A2:GDP. Una modificación única, la adición de etanolamina fosfoglicerol (EPG) a aminoácidos conservados (Glu301 y Glu374 en mamíferos), se ha observado aquí por primera vez. El análisis estructural de estos hallazgos facilita la comprensión de las múltiples funciones y regulaciones de eEF1A2. La adquisición de una muestra conformacionalmente homogénea de eEF1A2:GTP, necesaria para la unión a la plitidepsina, ha sido evaluada en ensayos de cristalización del complejo terciario de eEF1A2: GTP: plitidepsina. Con respecto al dominio de unión a la pared celular de la endolisina PlyP35 codificada por el fago P35 de Listeria monocytogenes (CBDP35), hemos resuelto la estructura cristalina de CBDP35 en un complejo con ácido teicoico natural de L. monocytogenes serovar 1/2a. Esta estructura es el primer módulo de unión a la pared celular en complejo con ácidos teicoicos jamás dilucidado. El análisis estructural reveló los principales determinantes para la unión de la pared celular bacteriana, en particular, el mecanismo molecular del reconocimiento de N-acetil-d-glucosamina, una decoración de carácter glicosídico en ácidos teicoicos de serovares patógenos de L. monocytogenes. Estos hallazgos arrojan luz sobre el desarrollo biotecnológico de nuevas herramientas en la industria alimentaria y las terapias derivadas de fagos para detectar y tratar infecciones bacterianas. / [CA] La cristal·lografia de raig X és una potent tècnica per a la resolució de l'estructura atòmica de macromolècules. La informació generada té gran impacte sobre diferents camps relacionats amb la investigació bàsica i aplicada, com són la biomedicina i disseny de fàrmacs, igual que en el desenvolupament d'aplicacions nanotecnológiques i biotecnològiques. Aquesta Tesi es centra en determinades problemàtiques actuals i en les proteïnes involucrades en les mateixes (TryR, eEF1A2 i CBDP35), sent estes subjecte de desenvolupament biotecnològic en els camps de la biomedicina, farmàcia i de la indústria alimentària, en el que la cristal·lografia de raig X juga un paper crucial per a dilucidar les seues estructures atòmiques i funcions. En consideració a la biomedicina i disseny de fàrmacs, hem resolt l'estructura de la Tripanotión reductasa (TryR) de Leishmania infantum en complex amb potents inhibidors de la seua activitat oxidorreductasa, amb potencial de desenrotllament com a fàrmacs. Així, s'ha caracteritzat la unió i mecanisme d'acció d'estos inhibidors. TryR és una reconeguda diana farmacològica per al tractament de la malaltia de Chagas, la Tripanosomiasi Humana Africana i la leishmaniosi, ja que exerceix un paper crucial i essencial en el metabolisme redox dels paràsits de la família Trypanosomatidae. A més, s'han analitzat els paràmetres de cristal·lització i difracció de nous inhibidors de la dimerizació de TryR, el disseny racional dels quals es basa en la unió a la interfície de dimerización de la mateixa. L'oncoproteína eEF1A2, involucrada en múltiples funcions cel·lulars i subjecte de nombroses modificacions posttraduccionals, s'unieix al fàrmac anticancerigen plitidepsina. La cristal·lografia de raig X, combinada amb experiments d'espectrometria de masses, s'han utilitzat com a ferramentes per a identificar noves modificacions posttraduccionals i característiques estructurals en eEF1A2:GDP. Una modificació única, l'addició d'etanolamina fosfoglicerol (EPG) a aminoàcids conservats (Glu301 i Glu374 en mamífers), s'ha observat ací per primera vegada. L'anàlisi estructural d'estes troballes facilita la comprensió de les múltiples funcions i regulacions d'eEF1A2. L'adquisició d'una mostra conformacionalmente homogènia d'eEF1A2:GTP, necessària per a la unió a la plitidepsina, ha sigut avaluada en assajos de cristal·lització del complex terciari d'eEF1A2: GTP: plitidepsina. Respecte al domini d'unió a la paret cel·lular de l'endolisina PlyP35 codificada pel fago P35 de Listeria monocytogenes (CBDP35), hem resolt l'estructura cristal·lina de CBDP35 en un complex amb àcid teicoico natural de L. monocytogenes serovar 1/2a. Esta estructura és el primer mòdul d'unió a la paret cel·lular en complex amb àcids teicoicos mai dilucidat. L'anàlisi estructural va revelar els principals determinants per a la unió de la paret cel·lular bacteriana, en particular, el mecanisme molecular del reconeixement de N-acetil-d-glucosamina, una decoració de caràcter glicosídico en àcids teicoicos de serovares patògens de L. monocytogenes. Estes troballes fan llum sobre el desenrotllament biotecnològic de noves ferramentes en la indústria alimentària i les teràpies derivades de fagos per a detectar i tractar infeccions bacterianes. / [EN] X-ray crystallography is a powerful technique for atomic structure resolution of macromolecules. The information generated impacts different fields involving basic and applied research on biomedicine and drug design and the development of nanotechnology and biotechnological applications. This dissertation focuses on current problematics and the target proteins involved (TryR, eEF1A2 and CBDP35) that are in sight for biotechnological development in the biomedical, pharmaceutical and food industry fields, in which X-ray crystallography plays a crucial role in the elucidation of their atomic structures and functions. Attaining to biomedical and drug design problematics, we have solved the structure of Leishmania infantum TryR in complex with potent oxidoreductase inhibitors prone to further development as anti-trypanosomal drugs, thereby characterizing their binding and mechanism of action. This protein is a long recognized drug target for the treatment of Chagas disease, Human African Trypanosomiasis and leishmaniasis, as it plays a crucial and essential role in the redox-metabolism of the Trypanosomatidae parasites. Moreover, the crystallization and diffraction parameters of novel TryR dimerization disruptors have been assayed for inhibitors which have been rationally designed to bind the dimerization interface of TryR. The "moonlighting" oncoprotein eEF1A2 is known to be highly post-translationally modified and to bind the anticancer drug plitidepsin. X-ray crystallography, combined with mass-spectrometry experiments, have been used as tools to identify novel post-translational modifications and structural features in eEF1A2:GDP. A unique modification, namely the addition of ethanolamine phosphoglycerol (EPG) to conserved glutamic residues (Glu301 and Glu374 in mammals), has been here observed for the first time. Structural analysis of these findings facilitate the understanding of eEF1A2's multiple functions and regulations. The acquirement of a conformationally homogenous eEF1A2:GTP sample, necessary for plitidepsin binding, has been has been assayed for eEF1A2:GTP:plitidepsin complex crystallization. Regarding the cell wall binding domain of Listeria monocytogenes phage-encoded endolysin PlyP35 (CBDP35), we have solved the crystal structure of CBDP35 in complex with natural Listeria serovar 1/2a teichoic acid. This structure is the first cell wall binding module in complex with teichoic acids ever elucidated. Structural analysis revealed the main determinants for bacterial cell-wall binding, in particular, the molecular mechanism of N-acetyl-d-glucosamine recognition, a glycosidic moiety in teichoic acids of pathogenic serovars of L. monocytogenes. These findings shed light upon the biotechnological development of new tools in the food industry and phage-derived therapies to detect and treat bacterial infections. / Agradecer al Ministerio de Educación, Cultura y Deporte por haberme proporcionado el contrato FPU (FPU14/03190) que me ha permitido desarrollar esta Tesis Doctoral en el Instituto de Química-Física “Rocasolano” del Consejo Superior de Investigaciones Científicas (IQFR-CSIC), así como la financiación otorgada para poder realizar mi estancia predoctoral en el laboratorio del Prof. Hammershmidt, en Greifswald, Alemania (EST17/00751). / Carriles Linares, AÁ. (2019). STRUCTURAL BIOMEDICINE: CHARACTERIZATION OF THE STRUCTURAL BASIS IN PROTEIN-DRUG RECOGNITION IN DIFFERENT HUMAN DISEASES [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/130844

Asiatic cholera has been called the scourge of the nineteenth century, for it caused the untimely death of millions throughout the world. During its four visits to the United States, unknown thousands of Kentuckians fell victims to the disease. In attempting to prevent the dreaded scourge, Kentuckians became more conscious of the need for cleaner cities, pure water and adequate sewage disposal. Modern waterworks facilities, sewage treatment and disposal facilities have provided the means by which the United States has conquered this scourge of the nineteenth century, for with these facilities cholera is the easiest of all communicable diseases to prevent. But, as with the eradication of any disease, constant vigilance and continued use of modern scientific knowledge are necessary to prevent its return. The disease is presently ravaging India and the Far East, and with modern jet travel it could bypass quarantine stations and enter the United States undetected. The “seeds” of the pestilence could be sown across the nation within a few hours. The only safeguard is modern sanitation facilities, for no permanent inoculation or miraculous cure has been developed. Today many rural areas of Kentucky and other states use wells and old cisterns that are, or could easily become, contaminated by human fecal matter. A fifth visit from cholera should not be necessary to correct the ignorance and complacent attitudes concerning inadequate sanitation facilities that exist in these areas of the nation. This study attempts to show the horrors of cholera’s four visits to Kentucky, and how the fear of the disease stimulated interest in public health.