Biology subjects

Garcia-Rodriguez, F.-J.

Publications and source records attributed to Garcia-Rodriguez, F.-J..

4 recordsLinked to original sources

bioRxiv2026

Single-cell metabolomics reveals infection-specific metabolic reprogramming of human macrophages

During intracellular infection, host cells adopt different metabolic states that traditional bulk analyses cannot distinguish. Using single-cell spatial metabolomics of human macrophages infected with Legionella pneumophila, we show that bacterial uptake activates host metabolism, whereas the bacterial effectors secreted through the type IV secretion system counteract this response and promote a glycolytic shift. The activity of effectors also generates distinct metabolic states within the infected macrophage population.

microbiology↗

bioRxiv2025

The Legionella effector RidL promotes mitochondrial fragmentation through phosphorylation activation of the large GTPase Drp1

Intracellular pathogens such as Legionella pneumophila secrete effector proteins that manipulate host cell processes to promote bacterial survival. One such effector, RidL, is known to inhibit retrograde trafficking by interacting with the retromer complex via its N-terminal domain. Here, we identify a second function of RidL mediated by its C-terminal domain, which directly binds to the mitochondrial fission GTPase Drp1 and related large GTPases. In vitro, RidL reduces Drp1 GTPase activity and disrupts its oligomerization. During infection, RidL localizes to mitochondria, enhances the accumulation of Drp1 and the outer membrane protein Tom20, and impairs mitochondrial dynamics and function. Moreover, in L. pneumophila-infected cells, RidL promotes phosphorylation of Drp1 at Ser616, leading to Drp1 activation and mitochondrial fragmentation. These findings establish RidL as a bifunctional effector that targets both the retromer complex and Drp1 through distinct domains. By interfering with host mitochondrial dynamics, RidL enables L. pneumophila to remodel host organelles and optimize conditions for intracellular replication.

microbiology↗

bioRxiv2025

Complex-I Preserves Mitochondrial Polarization during Infection of Human Macrophages by Secretion-competent Bacteria

Intracellular bacteria remodel host bioenergetics and modulate mitochondrial membrane potential ({Delta}{psi}m). However, how individual electron-transport chain (ETC) components sustain {Delta}{psi}m during infection of primary human macrophages remains unclear. Here we combine extracellular flux analysis with single-cell live imaging to understand how the ETC functions in human monocyte-derived macrophages (hMDMs) during infection with (Legionella pneumophila (Lp) or Salmonella enterica serovar Typhimurium (S.Tm). At 5 h post-infection, the Lp type IV secretion system (T4SS) and the S.Tm SPI-1 T3SS were required for the early drop of the oxygen consumption rate. Despite reduced respiration, the {Delta}{psi}m was preserved in all infection conditions and pathogen-specific strategies to maintain the {Delta}{psi}m were revealed. While Lp infection modulates the FOF1-ATPase to function in the reverse mode (hydrolase) with the adenine-nucleotide translocator (ANT) remaining in forward mode, S.Tm does not reverse the FOF1-ATPase during infection. Systematic inhibition of ETC complexes established that Complex I is uniquely required to maintain the {Delta}{psi}m during infection with virulent bacteria but not with secretion-deficient mutant strains. Complex II is required in all infection conditions but its inhibition had a minimal effect in non-infected cells, indicating infection-driven participation of this complex in the electron flow in the ETC coupled with the preservation of the {Delta}{psi}m. Complexes III and IV were essential in infected and non-infected cells. Together, our results identify a Complex I-driven maintenance of the {Delta}{psi}m, establishing Complex I as a bioenergetic checkpoint that distinguishes virulent from secretion-deficient intracellular bacteria. Furthermore we reveal that divergent strategies are employed by Lp and S.Tm to preserve macrophage mitochondrial polarization early during infection.

microbiology↗

bioRxiv2025

Provision of Preferred Nutrients to Macrophages Enables Salmonella to Replicate Intracellularly Without Relying on Type III Secretion Systems

Intracellular survival and replication within macrophages are key virulence determinants of Salmonella enterica serovar Typhimurium. This phenomenon is traditionally attributed to the activity of its two Type III Secretion Systems (T3SS) and their associated effectors. A critical challenge for these bacteria is acquiring nutrients from inside the host cell. Thus, they modulate the metabolism of host cells to replicate. Given the metabolic plasticity of macrophages, a key unresolved question is how their metabolic heterogeneity shapes intracellular Salmonella replication. By using human primary macrophages and live-cell imaging to monitor bacterial dynamics at the single-cell level, we revealed that Salmonella does not replicate in all infected cells. However, supplementation with specific carbon sources used by Salmonella during infection accelerated bacterial replication and increased the proportion of macrophages showing replicative bacteria. Remarkably, this occurred even in the absence of functional T3SSs, as a {Delta}prgH/{Delta}ssaV double mutant was able to replicate in a subset of infected cells under favorable nutrient conditions. These phenotypes are further amplified in macrophages with higher glycolytic activity, such as the murine RAW 264.7 cell line. Further analyses demonstrated that enhanced Salmonella replication is not strictly dependent on host glycolytic activity but is instead driven by the ability of the host cell to take up the nutrients Salmonella prefers for its replication early during infection. In summary, our findings suggest that the dependence of Salmonella on its T3SSs for intracellular replication can be bypassed when host cells provide optimal access to key nutrients and highlight the impact of metabolic heterogeneity in shaping intracellular bacterial replication during infection of macrophages.

microbiology↗