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Bacterial pathogens often deliver effectors into host cells using type 3 secretion systems (T3SS), the extremity of which forms a translocon that perforates the host plasma membrane. The T3SS encoded by Salmonella pathogenicity island 1 (SPI-1) is genetically associated with an acyl carrier protein, IacP, whose role has remained enigmatic. In this study, using tandem affinity purification, we identify a direct protein-protein interaction between IacP and the translocon protein SipB. We show, by mass spectrometry and radiolabelling, that SipB is acylated, which provides evidence for a modification of the translocon that has not been described before. A unique and conserved cysteine residue of SipB is identified as crucial for this modification. Although acylation of SipB was not essential to virulence, we show that this posttranslational modification promoted SipB insertion into host-cell membranes and pore-forming activity linked to the SPI-1 T3SS. Cooccurrence of acyl carrier and translocon proteins in several γ- and β-proteobacteria suggests that acylation of the translocon is conserved in these other pathogenic bacteria. These results also indicate that acyl carrier proteins, known for their involvement in metabolic pathways, have also evolved as cofactors of new bacterial protein lipidation pathways., Author Summary Acyl carrier proteins are small ubiquitous proteins involved in the synthesis of hydrocarbon based molecules. Notably, they are essential for the synthesis of fatty acids, which are the precursors of membrane phospholipids. They can also be involved in secondary metabolism, for example for the synthesis of molecules with antibacterial properties. Although acyl carrier proteins are widespread, the specific role of each individual protein seems comparatively poorly explored. In this study, we investigate the role of an acyl carrier protein genetically associated with a type 3 secretion system (T3SS). Many Gram-negative bacterial pathogens use T3SS to deliver effectors directly into the cytoplasm of eukaryotic host cells and to subvert host cellular pathways. For this purpose, the translocon, which is the terminal part of T3SS, forms a pore inserted into the host-cell membrane. Here we show that the acyl carrier protein associated with the T3SS has specialized to allow acylation of the translocon. The novel posttranslational modification of the translocon that we describe optimizes insertion into the host-cell membrane and pore-forming activity. This mechanism is likely to be conserved in other pathogenic bacteria given the conserved genetic association between T3SS and acyl carrier protein in several bacteria.

International audience; Iron–sulfur (Fe–S)‐containing proteins contribute to various biological processes, including redoxreactions or regulation of gene expression. Living organisms have evolved by developing distinct biosynthetic pathways to assemble these clusters, including iron sulfur cluster (ISC) and sulfur mobilization (SUF). Salmonella enterica serovar Typhimurium is an intracellular pathogen responsible for a wide range of infections, from gastroenteritis to severe systemic diseases. Salmonella possesses all known prokaryotic systems to assemble Fe–S clusters, including ISC and SUF. Because iron starvation and oxidative stress are detrimental for Fe–S enzyme biogenesis and because such environments are often met by Salmonella during its intracellular life, we investigated the role of the ISC and SUF machineries during the course of the infection. The iscU mutant, which is predicted to have no ISC system functioning, was found to be defective for epithelial cell invasion and for mice infection, whereas the sufBC mutant, which is predicted to have no SUF system functioning, did not present any defect. Moreover, the iscU mutant was highly impaired in the expression of Salmonella pathogenicity island 1 (Spi1) type III secretion system that is essential for the first stage of Salmonella infection. The Fe–S cluster sensor IscR, a transcriptional regulator matured by the ISC machinery, was shown to bind the promoter of hilD, which encodes the master regulator of Spi1. IscR was also demonstrated to repress hilD and subsequently Spi1 gene expression, consistent with the observation that an IscR mutant is hyperinvasive in epithelial cells. Collectively, our findings indicate that the ISC machinery plays a central role in Salmonella virulence through the ability of IscR to down‐regulate Spi1 gene expression. At a broader level, this model illustrates an adaptive mechanism used by bacterial pathogens to modulate their infectivity according to iron and oxygen availability.