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Structural determination of Streptococcus pyogenes M1 protein interactions with human immunoglobulin G using integrative structural biology.

ABSTRACT: Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen responsible for mild to severe, life-threatening infections. GAS expresses a wide range of virulence factors, including the M family proteins. The M proteins allow the bacteria to evade parts of the human immune defenses by triggering the formation of a dense coat of plasma proteins surrounding the bacteria, including IgGs. However, the molecular level details of the M1-IgG interaction have remained unclear. Here, we characterized the structure and dynamics of this interaction interface in human plasma on the surface of live bacteria using integrative structural biology, combining cross-linking mass spectrometry and molecular dynamics (MD) simulations. We show that the primary interaction is formed between the S-domain of M1 and the conserved IgG Fc-domain. In addition, we show evidence for a so far uncharacterized interaction between the A-domain and the IgG Fc-domain. Both these interactions mimic the protein G-IgG interface of group C and G streptococcus. These findings underline a conserved scavenging mechanism used by GAS surface proteins that block the IgG-receptor (Fc?R) to inhibit phagocytic killing. We additionally show that we can capture Fab-bound IgGs in a complex background and identify XLs between the constant region of the Fab-domain and certain regions of the M1 protein engaged in the Fab-mediated binding. Our results elucidate the M1-IgG interaction network involved in inhibition of phagocytosis and reveal important M1 peptides that can be further investigated as future vaccine targets.

SUBMITTER: Khakzad H

PROVIDER: S-EPMC7817036 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Structural determination of Streptococcus pyogenes M1 protein interactions with human immunoglobulin G using integrative structural biology.

Khakzad Hamed H Happonen Lotta L Karami Yasaman Y Chowdhury Sounak S Bergdahl Gizem Ertürk GE Nilges Michael M Tran Van Nhieu Guy G Malmström Johan J Malmström Lars L

PLoS computational biology 20210107 1

Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen responsible for mild to severe, life-threatening infections. GAS expresses a wide range of virulence factors, including the M family proteins. The M proteins allow the bacteria to evade parts of the human immune defenses by triggering the formation of a dense coat of plasma proteins surrounding the bacteria, including IgGs. However, the molecular level details of the M1-IgG interaction have remained unclear. Here, ...[more]

PMID: 33411763

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Project description:A total of 163 genes were found to be differentially expressed; 38 genes of them were up regulated and 125 genes were down regulated. Most notably the functional categories that were affected include Virulence genes (18 genes 11% of the total significantly differentiated genes), carbohydrate metabolism and cell envelop realted genes ( 31, 19.1% of the total), and aminoacid transport genes (21genes, 12.9%). Among the virulence-related genes, the most notable one were those belong to the production of capsule, the M protein, exotoxin, NAD glycohydrolase and Streptolysin S. Some of the capsule related genes were down regulated by more than 64 fold. These results indicate that CdhA (group A Streptococcal Cell division controlling and Chain-forming cell wall hydrolase) plays an important role in the regulation of virulence. S.pyogenes strain SF370 (wild-type) was procured from ATCC (ATCC 700294). M1-CdhA(-) mutant strain was obtained using pFW5 vector containing spectinomycin resistance marker (aad9). The mutant lacking CdhA is highly defective in cell division and growth, lacks antiphagocytic property and attenuated for virulence. Both strains (M1-Wild-type and M1-CdhA(-) were grown in Todd-Hewitt broth to their late log phase. Bacteria were harvested by centrifugation and washed twice with sterile PBS. Total RNA was isolated from these washed streptococci using Qiagen RNeasy Mini kit. For synthesis and labeling of cDNA, 20 ug of total RNA, 1.5 ug of random hexamer, 0.5mM dNTPs (except that 0.2mM of dTTP was replaced by the same amount of amino-allyl dUTP to incorporate dUTP into first-strand cDNA) were used in each reverse transcriptase reaction (Superscript II Reverse Transcriptase, Invitrogen). Purified cDNA preparations from the wild-type and M1CdhA(-) mutant strains were labeled with either Alexafluor-555 or Alexafluor-647 depending on the experimental design ( i.e. dye swap experiment). Differentially labeled probes were then combined and purified. Using three independently isolated RNA preparations (biological replicates), a total of 11 experiments (incorporating dye swaps) were performed. Accordingly eleven hybridization measurements for this mutant were performed. Thus Exp-1 to -4 (GSM388703, GSM388717, GSM388718 and GSM388719) are the technical replicates of the biological sample-1, Exp-5 to 8(GSM388732, GSM388733, GSM388734, and GSM388735) are technical replicates of biological sample-2 and Exp-9,-10, and -11 (GSM388736,GSM388737, and GSM388738) are technical replicates of the biological sample-3. Signals of the bound reagents on the microarray spots in terms of relative fluorescence values were measured and quantified by a laser scanner (GenePix 4100 ) at 10um/pixel resolution. The resulting images were processed using Gene Pix Pro software (version 4.0, Axon instruments). The raw data were obtained in the form of GenPix *.gpr output files. The web application CARMAweb (Comprehensive R based microarray analysis web service) was used for the normalization and analysis of microarray data. All raw data (in the form of *. GPR files) were uploaded to the web application in the data directory. Using appropriate navigation tree, background correction from the foreground signal was applied and within microarray normalization was achieved using the Lowess method. Genes flagged as bad spots by the scanning software were excluded from the analysis and all flagged spots were given a weight of zero. The normalized data were then subjected to fold-change analysis and t-statistics using Bioconductor multtest package. CARMAweb allows to set Log2 cut-off values for both the M (regulation) and the A (average expression) values. Differentially expressed genes (Log2 values of Mutant647-red vs. wild-type555-green or Log2 values Mutant-555(green) vs Wild-type 647-red) were defined based on cut-off value >1 Log2< i.e. all genes that show a two or more-fold up- or down-regulation. Bioconductors Multtest package provided suitable method to adjust P values according to multiple hypothesis testing problems.

Dataset Information

Structural determination of Streptococcus pyogenes M1 protein interactions with human immunoglobulin G using integrative structural biology.

Publications

Structural determination of Streptococcus pyogenes M1 protein interactions with human immunoglobulin G using integrative structural biology.

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