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Shigella are the causative agent of a disease called shigellosis. They carry a virulence plasmid, harboring genes encoding for a type three secretion system (T3SS) for invasion of human colonic epithelial cells. The T3SS is known to be responsible for injection of effector proteins into the host cells, triggering their uptake and facilitating the spread of the bacteria into adjacent cells. The T3SS machinery is encoded by a set of genes that are under the control of VirF and VirB transcriptional regulators. From a Transposon Directed Insertion Sequencing (TraDIS) screen, previously performed in our lab we identified four genes, ychQ, hyfR, padRlike and ycbU, where all showed a growth advantage when grown at 37°C, similar to virF and virB in this screen, suggesting a possible impact on the virulence. When Shigella are grown at 37°C the regulatory machinery for virulence is turned on, constituting a fitness cost for the bacteria. Therefore, we wanted to investigate on the impact of these four genes on the invasive behavior of Shigella. To do so, we have used a S. flexneri barcoded consortium, consisting of a mix of genetically tagged strains to perform in vitro fitness competition assays as well as barcoded infection assays. Here the mutants were allowed to compete with WT S. flexneri and ΔmxiD in a mixed consortium at 30°C (virulence off) or 37°C (virulence on). In these experiments we found that ΔychQ has a growth advantage compared to WT and ΔmxiD when grown at 37°C. Next, we also performed barcoded infection assays combined with gentamicin protection assays to analyze the invasive behavior of these mutants. Using a mixed consortium, consisting of seven tagged strains, in a single well set-up, we showed that none of the mutated genes were involved in invasion of Shigella. In summary, ychQ elicits a fitness cost when Shigella are grown at 37°C. However, we could not find an obvious link between fitness and host cell invasion. Instead, ychQ might likely be involved in some sort of thermoadaptation.