The Streptococcal Lipoprotein Rotamase A (SlrA) Is a Functional Peptidyl-prolyl Isomerase Involved in Pneumococcal Colonization

Abstract

Streptococcus pneumoniae expresses two surface-exposed lipoproteins, PpmA and SlrA, which share homology with distinct families of peptidyl-prolyl isomerases (PPIases). In this study, we demonstrated for the first time that the lipoprotein cyclophilin, SlrA, can catalyze the cis-trans isomerization of proline containing tetrapeptides and that SlrA contributes to pneumococcal colonization. The substrate specificity of SlrA is typical for prokaryotic and eukaryotic cyclophilins, with Suc-Ala-AlaPro-Phe-p-nitroanilide (pNA) being the most rapidly catalyzed substrate. In a mouse pneumonia model the slrA knock-out D39⌬slrA did not cause significant differences in the survival times of mice compared with the isogenic wild-type strain. In contrast, a detailed analysis of bacterial outgrowth over time in the nasopharynx, airways, lungs, blood, and spleen showed a rapid elimination of slrA mutants from the upper airways but did not reveal significant differences in the lungs, blood, and spleen. These results suggested that SlrA is involved in colonization but does not contribute significantly to invasive pneumococcal disease. In cell culture infection experiments, the absence of SlrA impaired adherence to pneumococcal disease-specific epithelial and endothelial non-professional cell lines. Adherence of the slrA mutant could not be restored by exogenously added SlrA. Strikingly, deficiency in SlrA did not reduce binding activity to host target proteins, but resulted in enhanced uptake by professional phagocytes. In conclusion, SlrA is a functional, cyclophilin-type PPIase and contributes to pneumococcal virulence in the first stage of infection, namely, colonization of the upper airways, most likely by modulating the biological function of important virulence proteins. Peptidyl-prolyl cis/trans isomerases (PPIases)6 are ubiquitous foldases, which accelerate the rate-limiting cis-trans or trans-cis conformational changes at Xaa-Pro bonds during protein folding in both eukaryotes and prokaryotes. There are three distinct families within the enzyme class of PPIases (EC 5.2.1.8): the cyclophilins, which bind the immunosuppressant cyclosporin A, the FK506-binding proteins, and the parvulins. The classical PPIase such as rotamase A of Escherichia coli is a member of the cyclophilin family (1, 2). The FK506-binding proteins all have high affinity for the immunosuppressant drug FK506 (3, 4). The ubiquitous bacterial trigger factor (5, 6) and the macrophage infectivity potentiator (Mip) protein of Legionella pneumophila (7) belong to the FK506-binding proteins. The parvulin family resembles enzymes such as parvulin, SurA, and PrsA. The E. coli parvulin 10 with its 92 amino acids is one of the smallest known PPIase enzymes and represents the prototype of this family (8). SurA contributes to the assembly of outer membrane proteins in Gram-negative bacteria (9). PrsA is considered to be involved in enzyme secretion and activation in Gram-positive bacteria (10 -12).Streptococcus pneumoniae (the pneumococcus) is a frequent colonizer of...

Lipoprotein biogenesis in Gram-positive bacteria: knowing when to hold ‘em, knowing when to fold ‘em

“…These lipoprotein PPIases are presumably well placed to interact with unfolded substrates emerging from the Sec translocon and could, thus, be important in processing virulence factors of Gram-positive pathogens. Streptococcus pneumoniae requires the SlrA PPIase for efficient murine colonisation [78] whereas a putative maturase lipoprotein mutant of S. equi was attenuated in both a mouse model and in a native equine host [47]. Finally, in staphylococci, the DsbA lipoprotein is a thiol-disulphide oxidoreductase apparently involved in disulphide bond formation in unidentified secreted protein substrates [79].…”

“…It is notable that several of these, including PrsA, belong to the parvulin subfamily of PPIases. However, because of sequence divergence, some representatives of this subfamily might not exhibit appreciable PPIase activity, in contrast to lipoprotein representatives of the cyclophilin family of PPIases [77,78]. These lipoprotein PPIases are presumably well placed to interact with unfolded substrates emerging from the Sec translocon and could, thus, be important in processing virulence factors of Gram-positive pathogens.…”

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

“…Consequently, pneumococci produce on the one hand proteins with manifold functions such as the adhesin PspC and, on the other hand, they are endowed with several proteins exhibiting similar functions which thus can compensate the defect of a protein due to mutations 4,17,18,35 . In these scenarios other in vivo infection experiments such as the coinfection approach has to be employed to decipher the effect of the loss-of-protein function in the mutant for colonization or invasive infections 27,28 .…”

“…5. Use a pipette with long narrow tips (Gel Loader Tips) and drop bioluminescent pneumococci in multiple small droplets onto the nares (10 µl/ nostril) of the mouse [26][27][28][29] . The mouse will involuntarily inhale the bacteria.…”

“…The findings have led to changes in in vivo virulence protocols. Instead of the inbred BALB/c mice often used in the past, the highly susceptible CD-1/MF1 outbred mouse strains are nowadays often used to study the effect of loss-of-function pneumococcal virulence or fitness factors [26][27][28] . Moreover, the availability of bioluminescent pneumococci and optical imaging techniques allows the real-time bioluminescence bioimaging of infections.…”