Mohammad Roghanian

Pseudomonas aeruginosa is a versatile, opportunistic pathogen, well-known for its adaptability to the challenging conditions encountered during infection of the human host. In chronic biofilm-associated infections, variations in motility and LPS structure are commonly observed adaptive traits. In this study, we identified differences in the expression of the O-specific antigen (OSA) biosynthetic cluster as a key driver of motility variation among a large number of clinical PA14 clade isolates. Despite retaining flagellar presence and function, OSA-deficient mutants exhibited increased hydrophobicity, which enhanced surface attachment and, in turn, reduced surface motility while promoting biofilm formation. The modified lipopolysaccharide structure with a lack of OSA also resulted in increased serum sensitivity, reduced phagocytic uptake, increased aminoglycoside resistance, and enhanced expression of the type III secretion system. These findings suggest that the commonly observed loss of OSA in P. aeruginosa isolates recovered from chronically infected cystic fibrosis lungs may be selectively advantageous. Importantly, we demonstrate that clinically relevant phenotypes influenced by the presence of OSA, such as motility and antibiotic resistance, become detectable only under test conditions that promote bacterial attachment. Similarly, the role of OSA expression in bacterial pathogenicity is conditional, with neither its presence nor its absence uniformly favoring infection. Together, our findings support a model in which OSA expression may be beneficial during early infection, whereas reduced OSA expression may promote traits associated with persistence at later stages. IMPORTANCE: Loss of the O-specific antigens (OSA) in Pseudomonas aeruginosa isolates recovered from chronically infected cystic fibrosis lungs is a commonly observed phenotype that may confer a selective advantage to this opportunistic pathogen. In this study, we demonstrate that P. aeruginosa strains lacking OSA become more adhesive, limiting their motility but enhancing biofilm formation. Furthermore, OSA-deficient strains exhibit increased serum sensitivity, while they are phagocytosed less, express elevated type III secretion system genes, and show enhanced antibiotic resistance. Our findings suggest that altered OSA expression may contribute to stage-dependent infection phenotypes, where reduced OSA expression during later stages drives biofilm formation and antibiotic resistance, thereby enabling persistence.

Antibiotic efficacy is often thought to depend not only on drug properties and bacterial susceptibility, but also on pathogen virulence and infection severity. However, during infection, virulence and bacterial growth are frequently confounded, making it difficult to determine whether virulence itself influences treatment outcome. Here, we used the Galleria mellonella larval infection model to disentangle the effects of virulence, in vivo growth, and antibiotic efficacy. We compared Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and Staphylococcus aureus across a wide range of inoculum sizes, revealing pronounced pathogen-specific differences in virulence. Despite these differences, all pathogens followed a similar infection trajectory, characterised by an initial reduction in bacterial burden followed by successful proliferation within the host. Focusing on specific Pseudomonas aeruginosa and Escherichia coli strains, which differ markedly in virulence, we identified conditions under which both exhibited comparable in vivo growth rates (doubling times of approximately 30 minutes), providing a controlled physiological framework to assess antibiotic efficacy independently of growth-related effects. Under these growth-matched conditions, treatment with ciprofloxacin or gentamicin at defined multiples of the MIC reduced bacterial loads, prevented host death, and cleared infections to a similar extent for both pathogens. These findings demonstrate that when bacterial growth is controlled in vivo, pathogen virulence does not determine antibiotic efficacy, supporting the predictive value of MIC-based susceptibility testing when bacteria are actively proliferating during infection.

Long RSH enzymes, Rel and RelA, are master regulators of bacterial (p)ppGpp alarmones levels. Bifunctional Rel transitions between a compact hydrolysis-competent (HD ^ON ) state, a relaxed catalytically inactive (HD ^OFF /SYNTH ^OFF ) state, and an elongated synthesis-competent (SYNTH ^ON ) state, whereas RelA samples only the latter two. The distribution of these states is controlled by starved ribosomes and regulatory proteins, including DarB, EIIA ^Ntr , ACP, NirD and YtfK. Here, we identify and characterize camelid nanobodies that act as selective allosteric modulators by stabilizing Rel and RelA in defined conformational states. Nanobodies that sequester the TGS domain of RelA prevent activation by deacylated tRNA on starved ribosomes, strongly inhibiting (p)ppGpp synthesis and suppressing Escherichia coli virulence in an animal model. Nb898 stabilizes Rel in the open SYNTH ^ON state, enhancing synthesis while suppressing hydrolysis, whereas Nb585 traps Rel in a hydrolysis-competent HD ^ON /SYNTH ^OFF conformation. Structural and biochemical analyses show that nanobodies, like endogenous allosteric regulators, restrict the conformational landscape of long RSH enzymes, establishing them as powerful tools for dissecting RSH function and as frameworks for developing protein-based RSH modulators.

Long RSH enzymes, Rel and RelA, are the master regulators of (p)ppGpp alarmone levels in bacteria. Their catalytic activity is governed by transitions between a compact, hydrolysis-competent (HD ^ON ) state and an elongated, synthesis-competent (SYNTH ^ON ) state. The equilibrium between these states is modulated by factors such as “starved” ribosomes and regulatory proteins DarB, EIIA ^NTR , ACP and YtfK. Here, we identify and characterize camelid nanobodies that act as selective allosteric modulators by trapping Rel/RelA enzymes in distinct conformational states. Nanobodies that lock the TGS domain of RelA and prevent its activation by deacylated tRNA on starved ribosomes, strongly inhibit (p)ppGpp synthesis and suppress the virulence of E. coli in an animal model. Nb898 stabilizes Rel in the open SYNTH ^ON state, enhancing synthesis activity while suppressing hydrolysis. Conversely, Nb585 traps Rel in a HD ^ON conformation, strongly inhibiting alarmone synthesis while promoting (p)ppGpp hydrolysis. Structural and biochemical analyses reveal that nanobodies, like natural allosteric regulators, act by restricting the RSH enzyme’s conformational landscape. These findings establish nanobodies as powerful tools for dissecting RSH function and provide potential leads for developing protein-based RSH modulators.

AlphaFold has revolutionized the decades-old issue of precisely predicting protein structures. However, its high accuracy relies on a computationally intensive step that involves searching vast databases for homologous sequences as the query protein of interest. Additionally, predicting the quaternary structure of protein complexes requires prior knowledge of subunit counts, a prerequisite rarely met. To address these limitations, we introduce FlashFold – a fast, user-friendly tool for protein structure prediction. It accelerates homology searches using a compact built-in database, enabling structure predictions up to 3-fold faster than AlphaFold3, with sacrificing little or no accuracy. Unlike others, FlashFold features adaptable built-in databases that allow users to easily incorporate their own privately sequenced data – an option that can positively influence the prediction accuracy. Moreover, it allows users to estimate stoichiometry of protein complexes directly from sequence information. To support high-throughput workflows and streamline downstream decision-making, it generates interactive and filterable summary reports, enabling users to efficiently visualize protein structures, and interpret large volumes of prediction results. FlashFold runs locally on Linux and Mac, eliminating reliance on third-party servers. FlashFold is available at https://github.com/chayan7/flashfold .

Stringent factors orchestrate bacterial cell reprogramming through increasing the level of the alarmones (p)ppGpp. In Beta- and Gammaproteobacteria, SpoT hydrolyzes (p)ppGpp to counteract the synthetase activity of RelA. However, structural information about how SpoT controls the levels of (p)ppGpp is missing. Here we present the crystal structure of the hydrolase-only SpoT from Acinetobacter baumannii and uncover the mechanism of intramolecular regulation of ‘long’-stringent factors. In contrast to ribosome-associated Rel/RelA that adopt an elongated structure, SpoT assumes a compact τ-shaped structure in which the regulatory domains wrap around a Core subdomain that controls the conformational state of the enzyme. The Core is key to the specialization of long RelA-SpoT homologs toward either synthesis or hydrolysis: the short and structured Core of SpoT stabilizes the τ-state priming the hydrolase domain for (p)ppGpp hydrolysis, whereas the longer, more dynamic Core domain of RelA destabilizes the τ-state priming the monofunctional RelA for efficient (p)ppGpp synthesis.

HflX is a ubiquitous bacterial GTPase that splits and recycles stressed ribosomes. In addition to HflX, Listeria monocytogenes contains a second HflX homolog, HflXr. Unlike HflX, HflXr confers resistance to macrolide and lincosamide antibiotics by an experimentally unexplored mechanism. Here, we have determined cryo-EM structures of L. monocytogenes HflXr-50S and HflX-50S complexes as well as L. monocytogenes 70S ribosomes in the presence and absence of the lincosamide lincomycin. While the overall geometry of HflXr on the 50S subunit is similar to that of HflX, a loop within the N-terminal domain of HflXr, which is two amino acids longer than in HflX, reaches deeper into the peptidyltransferase center. Moreover, unlike HflX, the binding of HflXr induces conformational changes within adjacent rRNA nucleotides that would be incompatible with drug binding. These findings suggest that HflXr confers resistance using an allosteric ribosome protection mechanism, rather than by simply splitting and recycling antibiotic-stalled ribosomes.

Summary: RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of Small Alarmone Synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analogue of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest employed by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2 and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3′ CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some Small Alarmone Hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as a novel class of RNA-modifying enzymes.

In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA’s CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a ‘closed’ conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an ‘open’ conformation, which primes its TGS and Helical domains for specific recognition and stabilization of cognate deacylated tRNA on the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity.