The double knockout also produced fewer (50%) plaques (Fig.?2B). Download Physique?S2, PDF file, 0.5 MB mbo005142039sf2.pdf (508K) GUID:?425D9CEA-76A1-48D2-8D5F-32F34DC22A16 Table?S1: Plasmids used in this study. Table?S1, PDF file, 0.03 MB. mbo005142039st1.pdf (33K) GUID:?2A18FA1D-A659-4E98-A645-4E3AA679A147 Table?S2: Primers used in this study. Table?S2, PDF file, 0.1 MB. mbo005142039st2.pdf (57K) GUID:?F96D56F0-1845-4E26-A8ED-4375C0E0A80E Chrysin ABSTRACT Host cell invasion by and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors around the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane segments, thus allowing the parasite to disengage from receptors and completely enter the host cell. To examine the specific roles of individual ROMs during invasion, we generated single, double, and triple knockouts for the three ROMs expressed in tachyzoites. Analysis of these mutants exhibited that ROM4 is the main protease involved in adhesin processing and host cell invasion, whereas ROM1 or ROM5 plays negligible functions in these processes. Deletion of ROM4 blocked the shedding of adhesins such as MIC2 (microneme protein 2), causing them to accumulate on the surface of extracellular parasites. Increased surface adhesins led to nonproductive attachment, altered gliding motility, impaired moving junction formation, and reduced invasion efficiency. Despite the importance of ROM4 for efficient invasion, mutants lacking all three ROMs were viable and MIC2 was still efficiently removed from the surface of invaded mutant parasites, implying the presence of ROM-independent mechanisms for adhesin removal during invasion. Collectively, these results suggest that although ROM processing of adhesins is not completely essential, it is important for efficient host cell invasion by express surface proteins that bind host cell receptors to aid invasion. Many of these adhesins are subject to cleavage by rhomboid proteases (ROMs) within their transmembrane segments during invasion. Previous studies have exhibited the importance of adhesin cleavage for parasite invasion and proposed that this ROMs responsible for processing would be essential for parasite survival. In and posterior localization around the parasite surface. TCF7L3 Here, we knocked out all three ROMs in tachyzoites and found that ROM4, but not ROM5, was important for adhesin cleavage. However, none of the ROMs individually or in combination was essential for cell access, further emphasizing that essential pathways such as invasion typically rely on redundant pathways to ensure survival. INTRODUCTION is an obligate intracellular pathogen infecting a wide range of animals as well as humans and can cause severe complications in immunocompromised individuals (1). Belonging to the phylum Apicomplexa, shares with other members of this large group of parasites a common set of structures and mechanisms for host cell invasion (2). For the apicomplexan parasites that are of clinical and veterinary significance, such as has six and has eight genes (23, 24). Of the six ROMs, ROM6 is usually predicted to be a mitochondrial PARL-like ROM Chrysin (14). The other five display stage-specific expression, with being expressed in tachyzoites; in bradyzoites; and in sporozoites (13, 14). Given that many adhesins important for tachyzoite invasion, such as MIC2 and AMA1, are substrates for ROMs (13), it is of great interest to know the exact roles of the different ROMs expressed at the tachyzoite stage. Previous work has resolved the role of individual ROMs in but has not established which, if any, of these proteases are essential. For example, ROM1 (TgROM1) localizes to micronemes, and knockdown of resulted in a mild growth defect (25). TgROM4 is usually evenly distributed around the plasma membrane (13), and conditional Chrysin suppression of this gene caused reduced adhesin shedding and decreased invasion (26). Additionally, overexpression of a dominant unfavorable mutant of inhibited AMA1 cleavage and blocked parasite replication after.
The double knockout also produced fewer (50%) plaques (Fig