Identifying Human Host Cell Protein Targets of the Bartonella Effector Protein (Bep) Fic Domains

<p>The genus Bartonellae represents an increasing number of emerging bacterial pathogens that utilises an unusual infection strategy, parasitising the red blood cells of their mammalian host. The most common species to infect humans are B. henselae and B. quintana. B. henselae is transmitted between cats by the cat flea, although occasionally infects humans via cat scratches or bites, causing cat-scratch disease (CSD). CSD is characterised by enlarged tender lymph nodes and fever. B. henselae also infects the endothelial cells of both its hosts; likely a factor in disease progression. B. quintana, the cause of trench fever during WWI, is spread people by the body louse. Trench fever is characterised by relapsing fever, headache, and bone pain. B. quintana is also able to infect human endothelial cells. These bacteria secrete a range of Bartonella effector proteins (Beps) via a Type IV secretion system, directly into endothelial cells, subverting host cell processes and resulting in internalisation of the bacteria. Beps have a range of functions, many of which are not fully characterised. B. henselae secretes three Beps (BepA-C) that contain a filamentation induced by cAMP (Fic) domain and a Bartonella Intracellular Delivery (BID) domain, with BepA being the best studied. BepA’s BID domain is responsible for intracellular delivery as well as inhibition of apoptosis by the host cell, however the exact function of the Fic domain remains unknown. Fic-containing bacterial toxins catalyse the transfer of an AMP moiety from ATP onto a host cell protein. This AMPylation frequently inactivates these proteins resulting in disrupted host cell processes and cytotoxicity. BepA has previously been shown to possess AMPylation activity, although the host target protein(s) are unknown. Evidence suggests that these proteins are novel targets. The aim of this study was to create protein constructs containing these Fic domains, and to develop techniques to identify the host cell target proteins post AMPylation. To this end, both a fluorescent ATP analogue and a fluorescent click chemistry based approach were utilised. While no target protein was identified, a basic methodology was developed for protein production and target protein identification that could be further developed.</p>

Identifying Human Host Cell Protein Targets of the Bartonella Effector Protein (Bep) Fic Domains

<p>The genus Bartonellae represents an increasing number of emerging bacterial pathogens that utilises an unusual infection strategy, parasitising the red blood cells of their mammalian host. The most common species to infect humans are B. henselae and B. quintana. B. henselae is transmitted between cats by the cat flea, although occasionally infects humans via cat scratches or bites, causing cat-scratch disease (CSD). CSD is characterised by enlarged tender lymph nodes and fever. B. henselae also infects the endothelial cells of both its hosts; likely a factor in disease progression. B. quintana, the cause of trench fever during WWI, is spread people by the body louse. Trench fever is characterised by relapsing fever, headache, and bone pain. B. quintana is also able to infect human endothelial cells. These bacteria secrete a range of Bartonella effector proteins (Beps) via a Type IV secretion system, directly into endothelial cells, subverting host cell processes and resulting in internalisation of the bacteria. Beps have a range of functions, many of which are not fully characterised. B. henselae secretes three Beps (BepA-C) that contain a filamentation induced by cAMP (Fic) domain and a Bartonella Intracellular Delivery (BID) domain, with BepA being the best studied. BepA’s BID domain is responsible for intracellular delivery as well as inhibition of apoptosis by the host cell, however the exact function of the Fic domain remains unknown. Fic-containing bacterial toxins catalyse the transfer of an AMP moiety from ATP onto a host cell protein. This AMPylation frequently inactivates these proteins resulting in disrupted host cell processes and cytotoxicity. BepA has previously been shown to possess AMPylation activity, although the host target protein(s) are unknown. Evidence suggests that these proteins are novel targets. The aim of this study was to create protein constructs containing these Fic domains, and to develop techniques to identify the host cell target proteins post AMPylation. To this end, both a fluorescent ATP analogue and a fluorescent click chemistry based approach were utilised. While no target protein was identified, a basic methodology was developed for protein production and target protein identification that could be further developed.</p>

Biopharmaceutical quality control with mass spectrometry

Mass spectrometry (MS) is a powerful technique for protein identification, quantification and characterization that is widely applied in biochemical studies, and which can provide data on the quantity, structural integrity and post-translational modifications of proteins. It is therefore a versatile and widely used analytic tool for quality control of biopharmaceuticals, especially in quantifying host-cell protein impurities, identifying post-translation modifications and structural characterization of biopharmaceutical proteins. Here, we summarize recent advances in MS-based analyses of these key quality attributes of the biopharmaceutical development and manufacturing processes.

Comparative analysis of ChAdOx1 nCoV-19 and Ad26.COV2.S SARS-CoV-2 vector vaccines

Vector-based SARS-CoV-2 vaccines have been associated with vaccine-induced thrombosis with thrombocytopenia syndrome (VITT/TTS), but the causative factors are still unresolved. We comprehensively analyzed ChAdOx1 nCov-19 (AstraZeneca) and Ad26.COV2.S (Johnson & Johnson). ChAdOx1 nCoV-19 contains significant amounts of host cell protein impurities, including functionally active proteasomes, and adenoviral proteins. In Ad26.COV2.S much less impurities were found. Platelet-factor 4 (PF4) formed complexes with ChAdOx1 nCoV-19 constituents, but not with purified virions from ChAdOx1 nCoV-19 or with Ad26.COV2.S. Vascular hyperpermeability was induced by ChAdOx nCoV-19 but not by Ad26.COV2.S.These differences in impurities together with EDTA-induced capillary leakage might contribute to the higher incidence rate of VITT associated with ChAdOx1 nCoV-19 compared to Ad26.COV2.S.

The Campylobacter jejuni CiaD effector co-opts the host cell protein IQGAP1 to promote cell entry

Campylobacter jejuni is a foodborne pathogen that binds to and invades the epithelial cells lining the human intestinal tract. Maximal invasion of host cells by C. jejuni requires cell binding as well as delivery of the Cia proteins (Campylobacter invasion antigens) to the host cell cytosol via the flagellum. Here, we show that CiaD binds to the host cell protein IQGAP1 (a Ras GTPase-activating-like protein), thus displacing RacGAP1 from the IQGAP1 complex. This, in turn, leads to the unconstrained activity of the small GTPase Rac1, which is known to have roles in actin reorganization and internalization of C. jejuni. Our results represent the identification of a host cell protein targeted by a flagellar secreted effector protein and demonstrate that C. jejuni-stimulated Rac signaling is dependent on IQGAP1.