Serotype M3 and M28 Group A Streptococci Have Distinct Capacities to Evade Neutrophil and TNF-α Responses and to Invade Soft Tissues

Abstract

The M3 Serotype of Group A Streptococcus (GAS) is one of the three most frequent serotypes associated with severe invasive GAS infections, such as necrotizing fasciitis, in the United States and other industrialized countries. The basis for this association and hypervirulence of invasive serotype M3 GAS is not fully understood. In this study, the sequenced serotype M3 strain, MGAS315, and serotype M28 strain, MGAS6180, were characterized in parallel to determine whether contemporary M3 GAS has a higher capacity to invade soft tissues than M28 GAS. In subcutaneous infection, MGAS315 invaded almost the whole skin, inhibited neutrophil recruitment and TNF-α production, and was lethal in subcutaneous infection of mice, whereas MGAS6180 did not invade skin, induced robust neutrophil infiltration and TNF-α production, and failed to kill mice. In contrast to MGAS6180, MGAS315 had covS G1370T mutation. Either replacement of the covS 1370T gene with wild-type covS in MGAS315 chromosome or in trans expression of wild-type covS in MGAS315 reduced expression of CovRS-controlled virulence genes hasA, spyCEP, and sse by >10 fold. MGAS315 covS wt lost the capacity to extensively invade skin and to inhibit neutrophil recruitment and had attenuated virulence, indicating that the covS G1370T mutation critically contribute to the hypervirulence of MGAS315. Under the background of functional CovRS, MGAS315 covS wt still caused greater lesions than MGAS6180, and, consistently under the background of covS deletion, MGAS6180 ΔcovS caused smaller lesions than MGAS315 ΔcovS. Thus, contemporary invasive M3 GAS has a higher capacity to evade neutrophil and TNF-α responses and to invade soft tissue than M28 GAS and that this skin-invading capacity of M3 GAS is maximized by natural CovRS mutations. These findings enhance our understanding of the basis for the frequent association of M3 GAS with necrotizing fasciitis.

Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of In Vitro and In Vivo Protease SpeB Expression and Virulence

“…The other new information regarding the virulence of RocA mutants from this study is that RocA mutants have a significantly lower capacity to cause local tissue damage, disseminate to organs, and inhibit neutrophil infiltration and are significantly less virulent than CovS mutants in subcutaneous infections in mice. These results are consistent with the attenuation of the virulence of hypervirulent, covS and rocA mutation-carrying serotype M3 strain MGAS315 by the replacement of the covS mutant gene with the wild-type covS gene (41). CovS and RocA mutations cause similar levels of enhancement of the expression of the same set of the virulence genes in vitro (20,24,42), and M122 (rocA C605A ) and M100 (covS Δ133TTCTT137 ) had similar high levels of expression of these virulence genes in vitro (Fig.…”

“…Large pieces of the remaining skin tissue were discarded, and there were about 4 ϫ 10 8 CFU bacteria in each sample according to the plating results. The bacteria from the in vitro cultures and murine skin were immediately processed to isolate total RNA, as described previously (41). RNA was then converted into cDNA using an All-in-One first-strand cDNA synthesis kit from GeneCopoeia (Rockville, MD).…”

The Mga Regulon but Not Deoxyribonuclease Sda1 of Invasive M1T1 Group A Streptococcus Contributes to In Vivo Selection of CovRS Mutations and Resistance to Innate Immune Killing Mechanisms

“…Plasmids pDCBBcovS and pDCBB-covR were used for in trans complementation of MGAS2221 covRS mutants selected in vivo. pDCBB-covS has been described (30), and pDCBB-covR was constructed as follows. The wildtype covR gene from MGAS2221 was PCR amplified using Phusion DNA polymerase (New England BioLabs, MA) and paired primers 5=-GGGGACAAGTTTGTACAAAAAAGCAGGCTAGAGGATAAGG GTTGGTATAA-3=/5=-GGGGACCACTTTGTACAAGAAAGCTGGG TTCCATATGACTTATTTCTCACGA-3=.…”

“…The production of M protein by GAS was assessed by Western blotting as described previously (28). Quantitative real-time reverse transcription-PCR (RT-PCR) analyses were performed using the All-in-One first-strand cDNA synthesis kit and the All-in-One SYBR quantitative PCR (qPCR) mix from GeneCopoeia (Rockville, MD), as described previously (30). Levels of mRNA for each target gene were normalized first to mRNA levels of the gyrA gene using the ⌬⌬C T method and then to those in MGAS2221.…”

Use of a Phosphorylation Site Mutant To Identify Distinct Modes of Gene Repression by the Control of Virulence Regulator (CovR) in Streptococcus pyogenes

“…Despite being studied for greater than 15 years, new information about the critical role of the CovRS system and its mechanistic principles continues to accumulate at a high rate (23)(24)(25)(26). It has recently been discovered that several regulatory pathways play a part in fine-tuning CovR phosphorylation status in response to different environmental cues (22-24, 27, 28).…”