Variations in the secondary structures of PAM proteins influence their binding affinities to human plasminogen

Qiu, Cunjia; Yuan, Yue; Zhong, Liang; Lee, Shaun W.; Ploplis, Victoria A.

doi:10.1016/j.jsb.2019.03.003

Cited by 16 publications

(17 citation statements)

References 44 publications

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“…As for virulence factors, temperature‐dependent changes in the expression levels of genes encoding the superantigen SpeA, EndoS glycosidase, α‐amylase and capsule amounts have also been reported (Xu and Collins, ; Nakamura et al , ; Kang et al , ). Furthermore, as a means of post‐translational thermoregulation of protein functions, temperature shift modulates the conformation of the M protein family and has effects on protein functions (Akerström et al , ; Qiu et al , ). Thus, it is likely that gene expressions, protein levels and protein functions in S. pyogenes are profoundly altered in response to changes in temperature.…”

Section: Discussionmentioning

confidence: 99%

Thermosensitive pilus production by FCT type 3 Streptococcus pyogenes controlled by Nra regulator translational efficiency

Nakata

Sumitomo

Patenge

et al. 2019

Molecular Microbiology

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Streptococcus pyogenes produces a diverse variety of pili in a serotype-dependent manner and thermosensitive expression of pilus biogenesis genes was previously observed in a serotype M49 strain.However, the precise mechanism and biological significance remain unclear. Herein, the pilus expression analysis revealed the thermosensitive pilus production only in strains possessing the transcriptional regulator Nra. Experimental data obtained for nra deletion and conditional nra-expressing strains in the background of an M49 strain and the Lactococcus heterologous expression system, indicated that Nra is a positive regulator of pilus genes and also highlighted the importance of the level of intracellular Nra for the thermoregulation of pilus expression. While the nra mRNA level was not significantly influenced by a temperature shift, the Nra protein level was concomitantly increased when the culture temperature was decreased. Intriguingly, a putative stem-loop structure within the coding region of nra mRNA was a factor related to the post-transcriptional efficiency of nra mRNA translation. Either deletion of the stemloop structure or introduction of silent chromosomal mutations designed to melt the structure attenuated Nra levels, resulting in decreased pilus production. Consequently, the temperature-dependent translational efficacy of nra mRNA influenced pilus thermoregulation, thereby potentially contributing to the fitness of nra-positive S. pyogenes in human tissues.

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Section: Discussionmentioning

confidence: 99%

Thermosensitive pilus production by FCT type 3 Streptococcus pyogenes controlled by Nra regulator translational efficiency

Nakata

Sumitomo

Patenge

et al. 2019

Molecular Microbiology

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“…4A show that hPg binds similarly to covS ϩ and srtA-C whole cells, showing that the levels of PAM on these cell surfaces are very similar. The data further show that isogenic strains in which the pam gene is inactivated (Δpam cells) interact with hPg to approximately 5% of the levels of the covS ϩ and SrtA-C cells, demonstrating that little binding of hPg occurs in the absence PAM and that PAM is the major tight-binding hPg protein on AP53 cells (10,48,49). In isogenic cells with a targeted inactivation of srtA, the PAM that is retained in the cytoplasmic membrane is nonetheless displayed on the GAS cell surface to ϳ75% of the PAM level in covS ϩ cells (Fig.…”

Section: Resultsmentioning

confidence: 77%

“…In this communication, we focus on pattern D strains of GAS, the subclass containing the largest number of emm types (6). As an example, we employ the prototype GAS AP53, a well-studied pattern D GAS strain (7)(8)(9)(10). M proteins from pattern D strains have the unique property of directly interacting with host human plasminogen (hPg), which is necessary for its pathogenicity, and are thus globally referred to as plasminogenassociated M (PAM) proteins.…”

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confidence: 99%

The M Protein of Streptococcus pyogenes Strain AP53 Retains Cell Surface Functional Plasminogen Binding after Inactivation of the Sortase A Gene

et al. 2020

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Streptococcus pyogenes (Lancefield group A Streptococcus [GAS]) is a β-hemolytic human-selective pathogen that is responsible for a large number of morbid and mortal infections in humans. For efficient infection, GAS requires different types of surface proteins that provide various mechanisms for evading human innate immune responses, thus enhancing pathogenicity of the bacteria. Many such virulence-promoting proteins, including the major surface signature M protein, are translocated after biosynthesis through the cytoplasmic membrane and temporarily tethered to this membrane via a type 1 transmembrane domain (TMD) positioned near the COOH terminus. In these proteins, a sorting signal, LPXTG, is positioned immediately upstream of the TMD, which is cleaved by the membrane-associated transpeptidase, sortase A (SrtA), leading to the covalent anchoring of these proteins to newly emerging l-Ala–l-Ala cross-bridges of the growing peptidoglycan cell wall. Herein, we show that inactivation of the srtA gene in a skin-tropic pattern D GAS strain (AP53) results in retention of the M protein in the cell membrane. However, while the isogenic AP53 ΔsrtA strain is attenuated in overall pathogenic properties due to effects on the integrity of the cell membrane, our data show that the M protein nonetheless can extend from the cytoplasmic membrane through the cell wall and then to the surface of the bacteria and thereby retain its important properties of productively binding and activating fluid-phase host plasminogen (hPg). The studies presented herein demonstrate an underappreciated additional mechanism of cell surface display of bacterial virulence proteins via their retention in the cell membrane and extension to the GAS surface. IMPORTANCE Group A Streptococcus pyogenes (GAS) is a human-specific pathogen that produces many surface factors, including its signature M protein, that contribute to its pathogenicity. M proteins undergo specific membrane localization and anchoring to the cell wall via the transpeptidase sortase A. Herein, we explored the role of sortase A function on M protein localization, architecture, and function, employing, a skin-tropic GAS isolate, AP53, which expresses a human plasminogen (hPg)-binding M (PAM) Protein. We showed that PAM anchored in the cell membrane, due to the targeted inactivation of sortase A, was nonetheless exposed on the cell surface and functionally interacted with host hPg. We demonstrate that M proteins, and possibly other sortase A-processed proteins that are retained in the cell membrane, can still function to initiate pathogenic processes by this underappreciated mechanism.

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“…A (His) 6 -tag was engineered into the reverse primer for PAM for further facile purification by Ni+-based affinity chromatography. The detailed steps have been described earlier ( 57 ).…”

Section: Methodsmentioning

confidence: 99%

Streptococcus co-opts a conformational lock in human plasminogen to facilitate streptokinase cleavage and bacterial virulence

Ayinuola

Brito-Robinson

Ayinuola

et al. 2021

Journal of Biological Chemistry

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Virulent strains of Streptococcus pyogenes (GAS) recruit host single-chain human plasminogen (hPg) to the cell surface - where in the case of Pattern D strains of GAS - hPg binds directly to the cells through a surface receptor, plasminogen-binding group A streptococcal M-protein (PAM). The coinherited Pattern D GAS-secreted streptokinase (SK2b) then accelerates cleavage of hPg at the R561-V562 peptide bond, resulting in the disulfide-linked two-chain protease, plasmin (hPm). hPm localizes on the bacterial surface, assisting bacterial dissemination via proteolysis of host defense proteins. Studies using isolated domains from PAM and hPg revealed that the A-domain of PAM binds to the hPg kringle-2 module (K2hPg), but how this relates to the function of the full-length proteins is unclear. Herein, we use intact proteins to show that the lysine binding site (LBS) of K2hPg is a major determinant of the activation-resistant T-conformation of hPg. The binding of PAM to the LBS of K2hPg relaxes the conformation of hPg, leading to a greatly enhanced activation rate of hPg by SK2b. Domain swapping between hPg and mPg emphasizes the importance of the Pg latent heavy chain (residues 1-561) in PAM binding and shows that while SK2b binds to both hPg and mPg, the activation properties of SK are strictly attributed to the serine protease domain (residues 562-791) of hPg. Overall, these data show that native hPg is locked in an activation-resistant conformation that is relaxed upon its direct binding to PAM, allowing hPm to form and provide GAS cells with a proteolytic surface.

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Variations in the secondary structures of PAM proteins influence their binding affinities to human plasminogen

Cited by 16 publications

References 44 publications

Thermosensitive pilus production by FCT type 3 Streptococcus pyogenes controlled by Nra regulator translational efficiency

Thermosensitive pilus production by FCT type 3 Streptococcus pyogenes controlled by Nra regulator translational efficiency

The M Protein of Streptococcus pyogenes Strain AP53 Retains Cell Surface Functional Plasminogen Binding after Inactivation of the Sortase A Gene

Streptococcus co-opts a conformational lock in human plasminogen to facilitate streptokinase cleavage and bacterial virulence

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