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Lynskey, N. N.

Publications and source records attributed to Lynskey, N. N..

3 recordsLinked to original sources

M1 protein expression determines niche-specific spread of Streptococcus pyogenes

Invasive infections with Streptococcus pyogenes account for significant mortality. Cell surface M protein is believed to be essential for S. pyogenes virulence, with critical roles in dissemination and immune evasion. In contrast, the role of secreted M protein in infection is less clear. Here, we assessed the role of M protein in streptococcal colonisation, persistence, and invasion in respiratory and soft tissue murine infection models, and report that while cell-surface M protein is required to establish nasopharyngeal infection, it does not drive invasive infection. Cell-surface M protein was important for binding of major host matrix proteins and growth in immune human blood, but soluble M protein was the predominant determinant of lymphatic survival, tissue spread, and systemic infection in mice. A mutant emm1 strain which only expresses secreted M protein exhibited enhanced survival in the lymph node niche and overall competitive advantage over the wildtype parent strain in co-infections. Absence of M protein at the bacterial cell surface led to enhanced hyaluronan binding, providing explanation for homing to the draining lymph node. Together, these findings highlight potential nuance in the role of M protein that may impact vaccine approaches targeting M protein. Author SummaryS. pyogenes is a significant cause of illness, both mild and lethal, worldwide. Despite this, there are currently no approved vaccines. Many vaccine candidates are based on the M protein, a surface expressed anti-phagocytic factor that is thought to be critical for the ability of S. pyogenes to evade the host immune system. The unexpected selection of an M1 protein mutant strain following in vivo soft tissue infection raised many questions for us. If M protein is meant to be the critical factor in avoiding immune killing, how was this strain able to persist? One explanation could be that, although the strain was negative for M1 protein on the bacterial surface, it still secreted a truncated version of the protein with potential inflammatory effects. Importantly, the strain was selected in the draining lymph node pointing to a niche-specific advantage. We showed that cell surface-associated M1 protein, although required to establish nasopharyngeal infection, was not required in soft tissue infection, and that expression of a soluble, truncated form of M1 protein gave a survival advantage over the wild-type strain expressing cell wall-associated M1 protein. Furthermore, the absence of cell surface M protein led to enhanced hyaluronan binding providing explanation for lymphatic niche homing.

microbiology↗

Methylome-dependent transformation of emm1 group A streptococci

Genetic intractability presents a fundamental barrier to the manipulation of bacteria, hindering advancements in microbiological research. Group A Streptococcus (GAS), a lethal human pathogen currently associated with an unprecedented surge of infections world-wide, exhibits poor genetic tractability attributed to the activity of a conserved type 1 restriction modification system (RMS). RMS detect and cleave specific target sequences in foreign DNA that are protected in host DNA by sequence-specific methylation. Overcoming this "restriction barrier" thus presents a major technical challenge. Here we demonstrate for the first time that different RMS variants expressed by GAS give rise to genotype-specific and methylome-dependent variation in transformation efficiency. Further, we show that the magnitude of impact of methylation on transformation efficiency elicited by RMS variant TRDAG, encoded by all sequenced strains of the dominant and upsurge-associated emm1 genotype, is 100-fold greater than for all other TRD tested and is responsible for the poor transformation efficiency associated with this lineage. In dissecting the underlying mechanism, we developed an improved GAS transformation protocol whereby the restriction barrier is overcome by addition of the phage anti-restriction protein Ocr. This protocol is highly effective for TRDAG strains including clinical isolates representing all emm1 lineages, and will expedite critical research interrogating the genetics of emm1 GAS, negating the need to work in an RMS-negative background. These findings provide a striking example of the impact of RMS target sequence variation on bacterial transformation, and the importance of defining lineage-specific mechanisms of genetic recalcitrance. ImportanceUnderstanding the mechanisms by which bacterial pathogens are able to cause disease is essential to enable the targeted development of novel therapeutics. A key experimental approach to facilitate this research is the generation of bacterial mutants, through either specific gene deletions or sequence manipulation. This process relies on the ability to transform bacteria with exogenous DNA designed to generate the desired sequence changes. Bacteria have naturally developed protective mechanisms to detect and destroy invading DNA, and these systems severely impede the genetic manipulation of many important pathogens, including the lethal human pathogen group A Streptococcus (GAS). Many GAS lineages exist, of which emm1 is dominant among clinical isolates. Based on new experimental evidence, we identify the mechanism by which transformation is impaired in the emm1 lineage and establish an improved and highly efficient transformation protocol to expedite the generation of mutants.

microbiology↗

Streptococcal superantigen-induced expansion of human tonsil T cells leads to altered T follicular helper cell phenotype, B cell death, and reduced immunoglobulin release

BackgroundStreptococcal pyrogenic exotoxin (SPE)A expression is epidemiologically linked to streptococcal tonsillo-pharyngitis and outbreaks of scarlet fever, although the mechanisms by which superantigens confer advantage Streptococcus pyogenes are unclear. S. pyogenes is an exclusively human pathogen. As the leukocyte profile of tonsil differs from peripheral blood, the impact of SPEA production on human tonsil cell function was investigated.\n\nMethodsHuman tonsil cells from routine tonsillectomy, were co-incubated with purified streptococcal superantigens or isogenic streptococcal culture supernatants, differing only in superantigen content. Tonsil cell proliferation was quantified by tritium-incorporation, and cell surface characteristics assessed by flow-cytometry. Soluble mediators were measured using ELISA and quantitative (q)RT-PCR was performed for immunoglobulin gene expression.\n\nResultsTonsil T cells proliferated in response to SPEA and demonstrated typical release of pro-inflammatory cytokines. When cultured in the absence of superantigen, tonsil preparations released large quantities of immunoglobulin over 7d. In contrast, marked B cell apoptosis and abrogation of total IgA, IgM, and IgG production occurred in the presence of SPEA and other superantigens. In SPEA-stimulated cultures, T follicular helper (TfH) cells showed a reduction in CXCR5 expression, but up-regulation of CD134 (OX40), CD278 (ICOS) and CD150 (SLAM) expression, indicative of a phenotypic change in the TfH population and associated with impaired chemotactic response to CXCL13.\n\nConclusionsSPEA and other superantigens cause dysregulated tonsil immune function, driving T cells from TFH to a proliferating phenotype, with resultant loss of B cells and immunoglobulin production, providing superantigen-producing bacteria with a likely survival advantage.

microbiology↗