Streptococcus pneumoniae secretion chaperones PrsA, SlrA, and HtrA are required for competence, antibiotic resistance, colonization, and invasive disease.

Streptococcus pneumoniae is a Gram-positive bacterium and a significant health threat with the populations most at risk being children, the elderly, and the immuno-compromised. To colonize and transition into an invasive infectious organism, S. pneumoniae secretes virulence factors that are translocated across the bacterial membrane and destined for surface exposure, attachment to the cell wall, or secretion into the host. The surface exposed protein chaperones PrsA, SlrA, and HtrA facilitate S. pneumoniae protein secretion; however, the distinct roles contributed by each of these secretion chaperones have not been well defined. Tandem Mass-Tagged Mass Spectrometry and virulence, adhesion, competence, and cell wall integrity assays were used to interrogate the individual and collective contributions of PrsA, SlrA, and HtrA to multiple aspects of S. pneumoniae physiology and virulence. PrsA, SlrA, and HtrA were found to play critical roles in S. pneumoniae host cell infection and competence, and the absence of each of these secretion chaperones significantly altered the S. pneumoniae secretome in distinct ways. PrsA and SlrA were additionally found to contribute to cell wall assembly and resistance to cell wall-active antimicrobials and were important for enabling S. pneumoniae host cell adhesion during colonization and invasive infection. These findings serve to further illustrate the pivotal contributions of PrsA, SlrA, and HtrA to S. pneumoniae protein secretion and virulence.

Type III secretion by Yersinia pseudotuberculosis is reliant upon an authentic N-terminal YscX secretor domain.

YscX was discovered as an essential part of the Yersinia type III secretion system about 20 years ago. It is required for substrate secretion and is exported itself. Despite this central role, its precise function and mode of action remain unknown. In order to address this knowledge gap, this present study refocused attention on YscX to build on the recent advances in the understanding of YscX function. Our experiments identified an N-terminal secretion domain in YscX promoting its secretion, with the first five codons constituting a minimal signal capable of promoting secretion of the signal less ß-lactamase reporter. Replacing the extreme YscX N-terminus with known secretion signals of other Ysc-Yop substrates revealed that the YscX N-terminal segment contains non-redundant information needed for YscX function. Further, both in cis deletion of the YscX N-terminus in the virulence plasmid and ectopic expression of epitope-tagged YscX variants again lead to stable YscX production but not type III secretion of Yop effector proteins. Mislocalisation of the needle components, SctI and SctF, accompanied this general defect in Yops secretion. Hence, a coupling exists between YscX secretion permissiveness and the assembly of an operational secretion system.

The type III secretion chaperone SctY may shield the hydrophobic export gate-binding C-terminus of its substrate SctX.

Gram-negative bacteria such as Aeromonas and Yersinia spp. have developed mechanisms to inhibit the immune defense of their host. Effector proteins are directly injected into the host cytoplasm from the bacterial cytosol via type III secretion systems (T3SSs), where they modulate the cytoskeleton and signaling of the cell. Assembly of, and secretion via, T3SSs is tightly regulated by a number of bacterial proteins, including SctX (AscX in Aeromonas), the secretion of which is essential for T3SS function. Here, crystal structures of AscX in complex with SctY chaperones from Yersinia or Photorhabdus spp. carrying homologous T3SSs are described. There are crystal pathologies in all cases, with one crystal form diffracting anisotropically and the other two exhibiting strong pseudotranslation. The new structures reveal that the positioning of the substrate is very similar on different chaperones. However, the two C-terminal SctX helices that cap the N-terminal tetratricopeptide repeat of SctY shift and tilt depending on the identity of the chaperone. Moreover, the C-terminus of the α3 helix of AscX exhibits an unprecedented kink in two of the structures. In previous structures, the C-terminus of SctX protrudes beyond the chaperone as a straight helix: a conformation that is required for binding to the nonameric export gate SctV but that is unfavorable for binary SctX-SctY complexes due to the hydrophobicity of helix α3 of SctX. A kink in helix α3 may allow the chaperone to shield the hydrophobic C-terminus of SctX in solution.