Abstract
Sporulation is an ancient developmental process that involves the formation of a highly resistant endospore within a larger mother cell. In the model organism Bacillus subtilis, sporulation-specific sigma factors activate compartment-specific transcriptional programs that drive spore morphogenesis. σG activity in the forespore depends on the formation of a secretion complex, known as the “feeding tube,” that bridges the mother cell and forespore and maintains forespore integrity. Even though these channel components are conserved in all spore formers, recent studies in the major nosocomial pathogen Clostridium difficile suggested that these components are dispensable for σG activity. In this study, we investigated the requirements of the SpoIIQ and SpoIIIA proteins during C. difficile sporulation. C. difficile spoIIQ, spoIIIA, and spoIIIAH mutants exhibited defects in engulfment, tethering of coat to the forespore, and heat-resistant spore formation, even though they activate σG at wildtype levels. Although the spoIIQ, spoIIIA, and spoIIIAH mutants were defective in engulfment, metabolic labeling studies revealed that they nevertheless actively transformed the peptidoglycan at the leading edge of engulfment. In vitro pull-down assays further demonstrated that C. difficile SpoIIQ directly interacts with SpoIIIAH. Interestingly, mutation of the conserved Walker A ATP binding motif, but not the Walker B ATP hydrolysis motif, disrupted SpoIIIAA function during C. difficile spore formation. This finding contrasts with B. subtilis, which requires both Walker A and B motifs for SpoIIIAA function. Taken together, our findings suggest that inhibiting SpoIIQ, SpoIIIAA, or SpoIIIAH function could prevent the formation of infectious C. difficile spores and thus disease transmission.
Highlights
A small subset of bacteria can survive adverse environmental conditions by forming a metabolically dormant cell-type known as an endospore [1,2,3]
To first determine if C. difficile spore development depends on the SpoIIQ and SpoIIIA proteins, we constructed targeted gene disruptions in the σF-regulated spoIIQ and σE-regulated spoIIIAA and spoIIIAH genes using the ClosTron gene knockout system (S4 Fig, [42])
Whereas uniform staining of FM4-64 around the entire forespore or the presence of DICbright spore compartments was observed in wildtype sporulating cells 73% of the time, FM4-64 staining of sporulating spoIIQ and spoIIIAH mutants was restricted to the curved membrane at the mother cell-forespore interface, and no differential interference contrast (DIC)-bright forespore compartments were observed in these mutants, indicative of an engulfment defect
Summary
A small subset of bacteria can survive adverse environmental conditions by forming a metabolically dormant cell-type known as an endospore (referred to as a “spore” hereafter) [1,2,3]. When C. difficile spores are ingested by susceptible hosts, they germinate in the gut and outgrow to form toxin-secreting vegetative cells [7,11,12]. While the toxins produced by C. difficile are responsible for the disease infection symptoms, spores are essential for this obligate anaerobe to transmit disease [6]. Spores complicate C. difficile infection clearance because they are resistant to many disinfectants and inert to antibiotics [4]. As a result, they can persist in the environment for long periods of time and facilitate C. difficile disease recurrence [12,14,15]. Despite the importance of spores to the pathogenesis of C. difficile, the molecular mechanisms underlying infectious spore formation remain largely uncharacterized
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