Key determinants of secretion specificity and endopilus stability are identified, revealing how minor sequence variations in conserved nanomachines drive functional adaptation to diverse environments.
Abstract The type III secretion system (T3SS) is a virulence mechanism commonly used by Gram-negative bacterial pathogens to deliver virulence proteins, known as effectors, into infected cells. The T3SS secretes a range of different substrates: first the needle subunits, then the translocon pore components and finally a pathogen-specific range of effector proteins. Each of these classes of substrates interacts with a corresponding class of bacterial chaperones, which are required for their efficient secretion. The requirement for these chaperones has been attributed to multiple functions, including preventing premature substrate activity, maintaining substrate stability in the bacterial cytoplasm and mediating substrate targeting and secretion hierarchy. Here, we bring together what is known about the function of T3SS chaperones in a range of different bacterial pathogens. Through analysis of the conservation of chaperone sequence and structure, we discuss how these proteins interact with and support the secretion of diverse substrates. Finally, we evaluate the extent to which chaperones are universally required for effector secretion.
ABSTRACT Conjugation machines constitute a large subfamily of the bacterial type IV secretion systems (T4SSs). In the absence of recipient cell contact, conjugation machines of gram-negative bacteria produce conjugative pili by extracting pilin subunits from an inner membrane (IM) pool. Upon pilus-mediated or direct contact with a target cell, conjugation systems transition to the recruitment of a cognate mobile element for intercellular DNA transfer. T4SS subunit requirements for pilus assembly and substrate transfer are nearly the same, except that VirD4 is required only for the latter. VirD4 ATPases (also termed type IV coupling proteins [T4CPs]) stimulate early DNA processing reactions and physically couple the DNA substrate to the T4SS channel. By visualizing fluorescently labeled pili, we identified marked differences in numbers, gross morphologies, and cellular organization of pili encoded by IncF F and IncN pKM101 plasmids. Remarkably, deletion of T4CP genes from both plasmids conferred hyperpiliation, a phenotype suppressed by complementation with the wild-type gene, but not alleles encoding variants defective in nucleotide binding/hydrolysis, or DNA substrate binding. In the pKM101 system, the T4CP plus TraK, a DNA processing factor that directs relaxosome assembly at the plasmid’s origin-of-transfer (oriT) sequence, also fully suppressed hyperpiliation even in the absence of oriT. We propose that the binding of a cognate DNA substrate (or certain relaxosome components) triggers the T4CP to suppress pilus production by blocking elongation, or stimulating retraction or release, which, in turn, transitions the T4SS from its default pilus-generating (mate-seeking) mode to the activated DNA-translocation (mating) machine. IMPORTANCE Mobile genetic elements (MGEs), often with cargoes of antibiotic resistance and virulence determinants, disseminate widely among bacteria through their encoded type IV secretion systems (T4SSs). Newly assembled T4SSs produce conjugative pili, which promote attachment to biotic and abiotic surfaces. Upon pilus-mediated or direct contact with a recipient cell, T4SSs recruit type IV coupling proteins (T4CPs), which, in turn, promote MGE transfer through the T4SS channel. Through visualization of conjugative pili by fluorescence microscopy, we demonstrate that pili encoded by two model plasmids, IncF F and IncN pKM101, display gross differences in morphologies and spatial organization on Escherichia coli cells, implying the evolution of distinct strategies for adherence and mating pair formation. We further report that engagement of T4CPs and bound MGE substrates with the T4SS blocks pilus accumulation. We propose that the T4CP plays a critical role in transitioning the T4SS from its pilus production to DNA transfer modes. Mobile genetic elements (MGEs), often with cargoes of antibiotic resistance and virulence determinants, disseminate widely among bacteria through their encoded type IV secretion systems (T4SSs). Newly assembled T4SSs produce conjugative pili, which promote attachment to biotic and abiotic surfaces. Upon pilus-mediated or direct contact with a recipient cell, T4SSs recruit type IV coupling proteins (T4CPs), which, in turn, promote MGE transfer through the T4SS channel. Through visualization of conjugative pili by fluorescence microscopy, we demonstrate that pili encoded by two model plasmids, IncF F and IncN pKM101, display gross differences in morphologies and spatial organization on Escherichia coli cells, implying the evolution of distinct strategies for adherence and mating pair formation. We further report that engagement of T4CPs and bound MGE substrates with the T4SS blocks pilus accumulation. We propose that the T4CP plays a critical role in transitioning the T4SS from its pilus production to DNA transfer modes.
