The role of the Rab32/BLOC3 host defence pathway in controlling Staphylococcus aureus infections

金黄色葡萄球菌是畜牧业和兽医学领域的一个新兴问题，我们最近的研究表明，牲畜可能是致病性金黄色葡萄球菌的一个来源(1)，有可能在人群中传播。我们最近在小鼠巨噬细胞中发现了一种阻止伤寒沙门氏菌存活的抗菌交通途径。该途径的核心成分是小GTPase Rab32及其鸟嘌呤核苷酸交换因子BLOC3（2,3）。我们的假设是，这一途径对金黄色葡萄球菌的杀伤也是至关重要的，它可能是不同金黄色葡萄球菌菌株的毒力和宿主特异性的原因。金黄色葡萄球菌可能已经进化出了中和这一途径的策略，从而获得了在特定宿主免疫细胞内存活的能力，从而增加了它们的致病潜力。在这个项目中，我们将测试上述假设，追求以下目标。我们将：1)测试一系列人类和动物源金黄色葡萄球菌菌株对RAB32/ block -3途径的敏感性。具体来说，我们将回答以下问题：1)毒力与RAB32/ block -3通路敏感性之间是否存在相关性？2)是否有任何分子证据可以解释易感性的差异？2)研究金黄色葡萄球菌与不同种类巨噬细胞的相互作用。具体来说，我们将回答以下问题：1)细菌是否主动侵入巨噬细胞？2)它们如何在巨噬细胞内存活？3)是否有宿主限制/特异性？如果有，限制/特异性是否取决于宿主或细菌因素？Baldassarre博士在粘附受体和肌动蛋白细胞骨架等领域拥有广泛的分子生物学专业知识。这些细胞系统对巨噬细胞吞噬和微生物杀灭至关重要。它们也是在金黄色葡萄球菌感染期间被劫持的系统。菲茨杰拉德教授是金黄色葡萄球菌致病性方面的专家，他使用一系列分子和基因组工具来了解人类和牲畜金黄色葡萄球菌感染的起源和演变。两名专业知识相互重叠和互补的导师将确保学生在整个项目中得到全面的指导。它还将保证在一系列技术方面的一流培训，例如：-微生物技术，包括分子生物学，细菌生理学，无菌技术，微生物的生长和培养；-分子技术，如PCR、克隆、基因敲除的创建（包括CRISPR/Cas9）；哺乳动物细胞培养和金黄色葡萄球菌感染；-显微镜技术，包括荧光宽视场和共聚焦显微镜，实时成像和3D重建。

S. aureus is an emerging problem in animal agriculture and veterinary medicine and our recent studies have shown how livestock could represent a source of pathogenic S. aureus (1) with the potential to spread in human populations.We have recently discovered an antimicrobial traffic pathway that prevents the survival of Salmonella Typhi, the agent of typhoid fever, in mouse macrophages. Central components of this pathway are the small GTPase Rab32 and its guanine nucleotide exchange factor BLOC3 (2,3).Our hypothesis is that this pathway is also critical for the killing of S. aureus and it may be responsible for the virulence and host-specificity of different S. aureus strains. S. aureus may have evolved strategies to neutralise this pathway gaining the ability to survive inside specific host immune cells and therefore increasing their pathogenic potential.In this project we will test the above hypothesis pursuing the following aims.We will: 1) Test a range of S. aureus strains, of both human and animal origin, for their sensitivity to the RAB32/BLOC-3 pathway. Specifically, we will answer the following questions: 1) Is there any correlation between virulence and the RAB32/BLOC-3 pathway sensitivity? 2) Is there any molecular evidence that can explain any difference in susceptibility?2) Study the interaction of S. aureus with macrophages from different species. Specifically, we will answer the following questions: 1) Do the bacteria actively invade the macrophages? 2) How they survive inside the macrophages? 3) Is there any host restriction/specificity? If so, is the restriction/specificity depending on host or bacterial factors?Dr Baldassarre has an extensive molecular biology expertise in fields such as adhesion receptors and actin cytoskeleton. Those are cellular systems crucial for macrophage phagocytosis and microbial killing. They are also systems hijacked during S. aureus infection. Prof Fitzgerald is an expert in S. aureus pathogenicity and uses a range of molecular and genomic tools to understand the origin and evolution of S. aureus infection in human and livestock.The presence of two supervisors with both overlapping and complementary expertise will ensure that the student will receive a full guidance throughout the project. It will also guarantee a first-class training in a range of techniques such as:- microbial techniques including molecular biology, bacterial physiology, sterile technique, growth and culture of microorganisms;- molecular techniques such as PCR, cloning, creation of gene knockouts (including CRISPR/Cas9); mammalian cell cultures and S. aureus infection;- microscopy techniques including fluorescent wide field and confocal microscopy, live imaging and 3D reconstruction.