Heterologous expression of pneumococcal virulence factor PspC on the surface of Lactococcus lactis confers adhesive properties

Abstract

Lactococcus lactis is a non-pathogenic bacterium that is used in the food industry but is also used as a heterologous host to reveal protein functions of pathogenic bacteria. The adhesin PspC from Streptococcus pneumoniae is a choline-binding protein that is non-covalently anchored to the bacterial cell wall. To assess the exclusive impact of pneumococcal surface protein C (PspC) on the interplay with its host we generated recombinant L. lactis producing a nisin-inducible and covalently anchored variant of PspC on the lactococcal cell surface. A translational fusion of the 59-end of pspC3.4 with the 39-end of hic (pspC11.4) was designed to decorate the surface of L. lactis with a chimeric PspC. The PspC3.4 part comprises the first 281 aa residues of PspC3.4, while the Hic sequence consists of the proline-rich and sortase-anchored domain. The results demonstrated that PspC is sufficient for adhesion and subsequent invasion of host epithelial cells expressing the human polymeric Ig receptor (hpIgR). Moreover, invasion via hpIgR was even more pronounced when the chimeric PspC was produced by lactococci compared with pneumococci. This study shows also for the first time that PspC plays no significant role during phagocytosis by macrophages. In contrast, recruitment of Factor H via the PspC chimer has a dramatic effect on phagocytosis of recombinant but not wild-type lactococci, as Factor H interacts specifically with the amino-terminal part of PspC and mediates the contact with phagocytes. Furthermore, L. lactis expressing PspC increased intracellular calcium levels in pIgR-expressing epithelial cells, thus resembling the effect of pneumococci, which induced release of Ca 2+ from intracellular stores via the PspC-pIgR mechanism. In conclusion, expression of the chimeric PspC confers adhesive properties to L. lactis and indicates the potential of L. lactis as a suitable host to study the impact of individual bacterial factors on their capacity to interfere with the host and manipulate eukaryotic epithelial cells. INTRODUCTIONStreptococcus pneumoniae (pneumococci) is the aetiological agent of community-acquired pneumonia, septicaemia and bacterial meningitis, all associated with high morbidity and mortality (Cartwright, 2002). Pneumococcal infections commence at the nasopharynx, where the bacteria reside as commensals without harming the host niche. Colonization of the respiratory mucosal epithelium is a prerequisite for invasive infections, which are most likely accompanied by dramatic changes in the nasopharyngeal physical barriers and the expression of pneumococcal virulence factors (Orihuela et al., 2004). The repertoire of uptake systems for essential nutrients enables pneumococci to adapt to and survive in different physiological conditions. More importantly, pneumococci have a variety of virulence factors facilitating adherence to host cells and evasion from the host immune system. Pneumococcal adherence to host cells is, similar to other Gram-positive pathogens including Staphylococcus aureus and Streptococcus...

The Choline-binding Protein PspC of Streptococcus pneumoniae Interacts with the C-terminal Heparin-binding Domain of Vitronectin

“…(41). The recombinant pGKK1 plasmid was used for the inducible extracellular expression of PspC in L. lactis (41). To generate heterologous L. lactis expressing other PspC subtypes, the desired pspC DNA fragments were amplified by PCR using the chromosomal DNA of strain S. pneumoniae ATCC33400 (PspC2) or D39 (PspC3).…”

“…Gene knockouts of pneumococcal transformants were verified by PCR and, where applicable, by flow cytometric analysis. (41). The recombinant pGKK1 plasmid was used for the inducible extracellular expression of PspC in L. lactis (41).…”

“…PspC expressed on the surface of the heterologous expression system L. lactis retains its biological activities (41). To assess the differences of PspC subtypes and the role of the number of R domains on binding of vitronectin, PspC2 and PspC3, respectively, were fused to the LPSTG cell wall-anchoring motif of PspC11.4, also known as Hic, and expressed on the surface of L. lactis (Fig.…”

Pneumococcal Adhesins PavB and PspC Are Important for the Interplay with Human Thrombospondin-1

“…Therefore, S. pneumoniae undergoes phase variations in which the polysaccharide capsule is mainly degraded (8,9). In this scenario the interaction of bacterial surface adhesins with soluble host proteins, host cellular receptors, or components of the ECM represents the major strategy essential to achieve a stable colonization (10,11). The epithelial ECM comprises the interstitial matrix between cells and the basal lamina upon the epithelial layer, which is composed of proteoglycans and fibrous proteins (among others fibronectin, collagen, elastins, and laminins) as well as matricellular proteins like vitronectin (Vn) or hTSP-1 (12)(13)(14)(15)(16).…”

Getting to the heart of the matter: Role ofStreptococcus mutansadhesin Cnm in systemic disease

“…One way to address this issue is to utilize a heterologous expression system and a successful strain for which there is precedent with Streptococcus proteins is Lactococcus lactis. [34][35][36][37][38] As a Gram-positive coccus, L. lactis shares many of the systems required for surface protein export and display and yet, as a dairy industry starter microorganism, it lacks capacity to interact strongly with human cells and tissues. 39 Consequently, L. lactis can serve as an excellent "blank canvas" with which gain of function can be explored following expression of a heterologous protein.…”