Unraveling the molecular determinants of the anti-phagocytic protein cloak of plague bacteria
PLOS PATHOGENS(2022)
Abstract
The pathogenic bacterium Yersina pestis is protected from macrophage engulfment by a capsule like antigen, F1, formed of long polymers of the monomer protein, Caf1. However, despite the importance of this pathogen, the mechanism of protection was not understood. Here we demonstrate how F1 protects the bacteria from phagocytosis. First, we show that Escherichia coli expressing F1 showed greatly reduced adherence to macrophages. Furthermore, the few cells that did adhere remained on the macrophage surface and were not engulfed. We then inserted, by mutation, an "RGDS" integrin binding motif into Caf1. This did not change the number of cells adhering to macrophages but increased the fraction of adherent cells that were engulfed. Therefore, F1 protects in two separate ways, reducing cell adhesion, possibly by acting as a polymer brush, and hiding innate receptor binding sites needed for engulfment. F1 is very robust and we show that E. coli expressing weakened mutant polymers are engulfed like the RGDS mutant. This suggests that innate attachment sites on the native cell surface are exposed if F1 is weakened. Single-molecule force spectroscopy (SMFS) experiments revealed that wild-type F1 displays a very high mechanical stability of 400 pN. However, the mechanical resistance of the destabilised mutants, that were fully engulfed, was only 20% weaker. By only marginally exceeding the mechanical force applied to the Caf1 polymer during phagocytosis it may be that the exceptional tensile strength evolved to resist the forces applied at this stage of engulfment. Author summaryMacrophages, a type of white blood cell, form an important element of our immune defence. They interrogate other cells' surfaces for molecular clues and ingest those presenting a threat in a process known as phagocytosis. Not surprisingly, pathogenic bacteria have developed ways to evade this fate. The plague bacterium, Yersinia pestis, produces the long polymeric F1 coat protein which enables it to avoid ingestion, but the mechanism was unclear. We show that equipping Escherichia coli cells with an F1 coat protected them from phagocytosis by two separate mechanisms, reducing contact with the macrophage surface and hiding the signals that tell the macrophages they are targets. F1 is also a very stable protein polymer and using single molecule force spectroscopy we showed it also has a very high resistance to pulling forces. Surprisingly, mutations which reduced this by only 20% caused adherent bacteria to be fully ingested, indicating that cells are subject to significant forces prior to recognition and ingestion. Thus, F1 has evolved three notable properties (i) physical; creation of a hydrated polymer brush to inhibit surface interactions, (ii) chemical; absence of molecular recognition clues needed for engulfment and (iii) mechanical; strength that maintains the camouflage layer during surface stretching.
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Key words
plague bacteria,protein,anti-phagocytic
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