Biology subjects

Frando, A.

Publications and source records attributed to Frando, A..

5 recordsLinked to original sources

bioRxiv2026

Regulation of interbacterial interactions between Pseudomonas aeruginosa and Stenotrophomonas maltophilia by pqs quorum sensing

Pseudomonas aeruginosa, an opportunistic pathogen, uses a trio of quorum sensing (QS) systems to regulate the production of some virulence factors. Two of these, the las and rhl systems, involve acyl-homoserine lactone signals; the third, called pqs, primarily uses the signal 2-heptyl-3-hydroxy-4(1H)-quinolone ("PQS"). We aimed to identify how interbacterial interactions are regulated between P. aeruginosa and Stenotrophomonas maltophilia, which co-occur in many environments, including the airways of people with cystic fibrosis. We explored P. aeruginosa and S. maltophilia interactions using a co-culture model. In the conditions of our experiments, P. aeruginosa kills S. maltophilia. Co-culture of S. maltophilia with P. aeruginosa deficient in las, rhl, or pqs QS resulted in greater S. maltophilia viability than co-culture with the wildtype. This inhibition was not generally attributable to las and rhl-regulated toxins. Therefore, we interrogated the role of pqs QS and found that co-culture of S. maltophilia with P. aeruginosa deficient in PQS biosynthesis showed similar CFUs to monoculture. Exogenous PQS did not complement this phenotype, suggesting that another quinolone is the effector. We found that S. maltophilia killing is reduced in competition with a mutant that cannot make the quinolone HQNO (2-heptyl-4-quinoline N-oxide). We show that full killing of S. maltophilia by P. aeruginosa requires three components: HQNO, the chaperone PqsE, and intact PQS biosynthesis that together form a tripartite AND logic gate. Our work identifies quinolone quorum sensing as a driver for interactions between the Gram-negative pathogens P. aeruginosa and S. maltophilia.

microbiology↗

bioRxiv2025

PE/PPE proteins contribute to Mycobacterium tuberculosis drug resistance

The outer membrane (OM) of mycobacteria is a formidable permeability barrier that confers drug tolerance, and whether drugs traverse the OM by mechanisms beyond passive diffusion remains unclear. The proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) proteins of pathogenic mycobacteria include several OM transporters. Here, we tested the role of PE/PPE proteins in Mycobacterium tuberculosis (Mtb) drug transport and resistance. Mutations in multiple pe/ppe genes were strongly associated with drug resistance in a genetic association study, and mutations in ppe42 and ppe51 also conferred increased resistance in vitro. Deletion of a pe/ppe pair transcriptionally responsive to drug exposure, pe25/ppe41, led to elevated resistance to isoniazid (INH) across all major Mtb lineages and accelerated INH resistance emergence in vitro. These data identify a role for Mtb PE/PPE proteins in drug resistance consistent with the PE/PPE transporter paradigm, suggest a broader contribution of this large protein family, and a new factor of Mtb clinical drug resistance.

microbiology↗

bioRxiv2025

Modulation of the Pseudomonas aeruginosa quorum sensing cascade by MexT-regulated factors

Pseudomonas aeruginosa (Pa) uses quorum sensing (QS), a cell-cell communication system that enables it to sense cell density and to alter gene expression. Pa has three complete QS circuits controlled by the regulators LasR, RhlR, and PqsR, that together activate hundreds of genes. In the well-studied strain PAO1, QS is organized hierarchically, with PqsR and RhlR activity dependent on LasR. This hierarchy depends on the non-QS transcription factor MexT; deletion of mexT allows for RhlR activity in the absence of LasR. We aimed to identify how MexT modulates the Pa QS architecture. We compared the transcriptome of PAO1 to that of PAO1{Delta}mexT and determined a MexT regulon. We identified two MexT-regulated operons that may affect the QS hierarchy: the efflux pump genes mexEF-oprN and the Pseudomonas quinolone signal (PQS) synthesis genes pqsABCDE. We tested whether the products of these genes affected the QS hierarchy. A mexEF knockout mutant, like a mexT deletion mutant, exhibited RhlR activity earlier, and to a higher magnitude, than wild-type PAO1. MexEF-OprN is known to export quinolones, and we found that exogenous addition of PQS, through PqsE, also resulted in earlier and higher magnitude of RhlR activity compared to wild-type PAO1. We also discovered alternate QS architectures in clinical isolates, where RhlR activity is not fully dependent on LasR. In these isolates, surprisingly, MexT does not influence the relationship between LasR and RhlR. Our work reveals a new suite of factors that regulate QS in Pa, with implications for bacterial behaviors in environmental and clinical settings. ImportanceBacteria interact with both abiotic and biotic factors in their environment. Quorum sensing (QS) is one mechanism that bacteria use to communicate with other bacteria and coordinate behaviors in the population. QS regulates a wide variety of processes ranging from the production of light to modulation of virulence factors; some bacteria use single QS circuits, while others have several. The opportunistic pathogen Pseudomonas aeruginosa uses QS to control some virulence functions and has three complete QS circuits. Our study explores why bacteria might have multiple QS circuits. We show how a non-QS transcription factor, MexT, influences QS regulators in P. aeruginosa and we uncover the diversity of QS architectures in clinical isolates. This study begins to reveal the benefits (or disadvantages) of multiple QS circuits, allowing us to understand behaviors of bacteria that have a range of implications including in health, agriculture, and bioremediation.

microbiology↗

bioRxiv2022

Calcium transport by the Mycobacterium tuberculosis PE15/PPE20 proteins

Many aspects of eukaryotic physiology are regulated by calcium ions (Ca2+). Whether bacteria have similar Ca2+ systems for transport, storage, binding, and response to Ca2+ is not well understood. To identify components of Ca2+ signaling in Mycobacterium tuberculosis, we determined its transcriptional response to Ca2+. Overall, only few genes changed expression, suggesting a limited role of Ca2+ as a transcriptional regulator. However, two of the most strongly downregulated genes were the pe15 and ppe20 genes that code for members of a large family of proteins that localizes to the outer membrane. PE15 and PPE20 formed a complex and PPE20 directly bound Ca2+. Ca2+-associated phenotypes such as an increase in ATP consumption and increase in biofilm formation were reversed in a pe15/ppe20 knockout strain, suggesting a direct role in Ca2+ homeostasis. To test whether the complex has a role in Ca2+ transport across the outer membrane, we created a fluorescence resonance energy transfer (FRET)-based Ca2+ reporter strain. A pe15/ppe20 knockout in the FRET background showed a specific and selective loss of Ca2+ influx that was dependent on the presence of an intact outer cell wall. These data show that PE15/PPE20 form a Ca2+-binding protein complex that selectively imports Ca2+ and support the emerging idea of a general family-wide role of PE/PPE proteins in transport across the outer membrane.

microbiology↗

bioRxiv2022

The Mycobacterium tuberculosis protein O-phosphorylation landscape

Bacterial phosphosignaling has long been synonymous with the histidine kinases of the two component systems, but many bacteria, including Mycobacterium tuberculosis (Mtb), also code for Ser/Thr protein kinases (STPKs). STPKs are the main phosphosignaling enzymes in eukaryotes, but the full extent of phosphorylation on protein Ser/Thr and Tyr (O-phosphorylation) in bacteria remains unclear. Here, we explored the global signaling capacity of the STPKs in Mtb. We generated STPK loss-and gain-of-function strains and measured the resulting O-phosphorylation and transcriptional changes. This deep phosphoproteome shows that O-phosphorylation in Mtb is an underexplored protein modification that affects >70% of the proteome. The substrate-STPK interactions show an extensive interface with the transcriptional machinery, resulting in regulation of gene expression of [~]30% of Mtb genes. Mtb O-phosphorylation gives rise to an expansive, distributed, and cooperative network of a complexity that has previously only been associated with eukaryotic phosphosignaling networks.

microbiology↗