African Salmonella Typhimurium sequence type 313 lineage 2 evades MAIT cell recognition by overexpressing RibB

Mucosal-associated invariant T (MAIT) cells are a subset of innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defence. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with a selective absence of MAIT cells have not been identified. We report that typhoidal and non-typhoidal S. enterica strains generally activate MAIT cells. However, African invasive disease-associated multidrug-resistant S. Typhimurium sequence type 313 lineage 2 strains escape MAIT cell recognition through overexpression of ribB, a bacterial gene that encodes the 4-dihydroxy-2-butanone 4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. This MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that ribB overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of S. Typhimurium sequence type 313 lineage 2 in vivo.


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Identification of cellular responses to multiple Salmonella enterica subsp 119 enterica serovars.

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To identify potential differences in the response of innate and adaptive T cells to 122 distinct Salmonella pathovars, we focused on two pathovariants of S. Typhimurium

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indicating that the lack of cell activation is exclusive to MAIT cells (Fig 2A, 2C

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Paratyphi A strain; in addition two differently sourced stocks of STM-D23580 were 186 tested, to ensure that genuine sequence type 313 isolates were being used. At two different MOIs, STM-D23580 elicited the lowest levels of MAIT cell activation of the 188 group (Fig S1C-S1D). In contrast, gd T cell responses were comparable across all 189 Salmonella pathovars (S1E-S1F).

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We next determined whether the lack of MAIT cell activation was caused by the entire 192 S. Typhimurium sequence type 313 clade or was a unique characteristic of sequence 193 type 313 lineage 2 which is currently causing most clinical disease in Africa [14]. STM-

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D23580 and additional isolates of sequence type 313 lineage 2, were compared with 195 closely-related isolates that were members of sequence type 313 lineage 1 or the UK-196 sequence type 313 group that is associated with gastroenteritis ( Figure 2E). To

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In the case of bacterial supernatants, these were taken from late exponential phase 514 cultures, grown from a single colony following 18 hours incubation under constant 515 shaking. Supernatants were filter sterilised before using.

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To construct pPL-ribB, the ribB gene was amplified from genomic DNA of S.

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Typhimurium 4/74 using primers ribB_FW and ribB_RV. The PCR product was used 520 for a linear amplification reaction with plasmid pJV300 (pPL) using Phusion DNA 521 polymerase (New England Biolabs), and the resulting product was digested with DpnI.

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The plasmid was transformed into E. coli TOP10 and selected on LB plates

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After obtaining appropriate consent and medical authorisation, a peripheral blood 546 sample was collected between days 2 and 9 after positive blood culture. Aliquots of 547 whole blood were treated with Fix/lyse buffers (SmartTube) and stored frozen until use.

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For analysis, cases were divided in invasive nontyphoidal (iNTS) or typhoidal 549 Salmonellosis based on microbiology testing. As part of the same study, healthy adults 550 from the local community were enrolled as controls. A peripheral blood sample was 551 obtained from these individuals and processed in the same way as the cases. Since

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One aliquot of fixed/lysed whole blood was defrosted and washed three times in FACS 597 buffer. Cells were stained for surface and intracellular markers as described above and