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Journal articleLarrouy-Maumus GJ, 2019,
Lipids as biomarkers of cancer and bacterial infections
, Current Medicinal Chemistry, Vol: 26, Pages: 1924-1932, ISSN: 0929-8673Lipids are ubiquitous molecules, known to play important roles in various cellular processes. Alterations to the lipidome can therefore be used as a read-out of the signs of disease, highlighting the importance to consider lipids as biomarkers in addition of nucleic acid and proteins. This mini-review exposes the current knowledge and limitations of the use of lipids as biomarkers of the top global killers which are cancer and bacterial infections.
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Journal articleFrankel G, Schroeder GN, 2019,
The Galleria mellonella Infection Model for Investigating the Molecular Mechanisms of Legionella Virulence.
, Methods Mol Biol, Vol: 1921, Pages: 333-346Legionella species evolved virulence factors to exploit protozoa as replicative niches in the environment. Cell culture infection models demonstrated that many of these factors also enable the bacteria to thrive in human macrophages; however, these models do not recapitulate the complex interactions between macrophages, lung epithelial, and additional immune cells, which are crucial to control bacterial infections. Thus, suitable infection models are required to understand which bacterial factors are important to trigger disease. Guinea pigs and, most frequently, mice have been successfully used as mammalian model hosts; however, ethical and economic considerations impede their use in high-throughput screening studies of Legionella isolates or small molecule inhibitors.Here, we describe the larvae of the lepidopteran Galleria mellonella as insect model of Legionella pathogenesis. Larvae can be obtained from commercial suppliers in large numbers, maintained without the need of specialized equipment, and infected by injection. Although lacking the complexity of a mammalian immune system, the larvae mount humoral and cellular immune responses to infection. L. pneumophila strain 130b and other prototype isolates withstand these responses and use the Defective in organelle trafficking/Intracellular multiplication (Dot/Icm) type IV secretion system (T4SS ) to inject effectors enabling survival and replication in hemocytes, insect phagocytes, ultimately leading to the death of the larvae. Differences in virulence between L. pneumophila isolates or gene deletion mutants can be analyzed using indicators of larval health and immune induction, such as pigmentation, mobility, histopathology, and survival. Bacterial replication can be measured by plating hemolymph or by immunofluorescence microscopy of isolated circulating hemocytes from infected larvae. Combined, these straightforward experimental readouts make G. mellonella larvae a versatile model host to rapidly assess the v
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Journal articleSo EC, Mousnier A, Frankel G, et al., 2019,
Determination of In Vivo Interactomes of Dot/Icm Type IV Secretion System Effectors by Tandem Affinity Purification.
, Methods Mol Biol, Vol: 1921, Pages: 289-303The Dot/Icm type IV secretion system (T4SS) is essential for the pathogenesis of Legionella species and translocates a multitude of effector proteins into host cells. The identification of host cell targets of these effectors is often critical to unravel their roles in controlling the host. Here we describe a method to characterize the protein complexes associated with effectors in infected host cells. To achieve this, Legionella expressing an effector of interest fused to a Bio-tag, a combination of hexahistidine tags and a specific recognition sequence for the biotin ligase BirA, are used to infect host cells expressing BirA, which leads to biotinylation of the translocated effector. Following chemical cross-linking, effector interactomes are isolated by tandem affinity purification employing metal affinity and NeutrAvidin resins and identified by western blotting or mass spectrometry.
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Book chapterCosta TRD, Francis MK, Farag SI, et al., 2019,
Measurement of Yersinia Translocon Pore Formation in Erythrocytes
, Methods in Molecular Biology, Publisher: Springer New York, Pages: 211-229, ISBN: 9781493995400 -
Journal articleJewell P, Dixon L, Singanayagam A, et al., 2019,
Severe disseminated infection with emerging lineage of methicillin-sensitive Staphylococcus aureus
, Emerging Infectious Diseases, Vol: 25, Pages: 187-187 -
Journal articleKrokowski S, Lobato-Marquez D, Chastanet A, et al., 2018,
Septins recognize and entrap dividing bacterial cells for delivery to lysosomes
, Cell Host and Microbe, Vol: 24, Pages: 866-874, ISSN: 1931-3128The cytoskeleton occupies a central role in cellular immunity by promoting bacterial sensing and antibacterial functions. Septins are cytoskeletal proteins implicated in various cellular processes, including cell division. Septins also assemble into cage-like structures that entrap cytosolic Shigella, yet how septins recognize bacteria is poorly understood. Here, we discover that septins are recruited to regions of micron-scale membrane curvature upon invasion and division by a variety of bacterial species. Cardiolipin, a curvature-specific phospholipid, promotes septin recruitment to highly curved membranes of Shigella, and bacterial mutants lacking cardiolipin exhibit less septin cage entrapment. Chemically inhibiting cell separation to prolong membrane curvature or reducing Shigella cell growth respectively increases and decreases septin cage formation. Once formed, septin cages inhibit Shigella cell division upon recruitment of autophagic and lysosomal machinery. Thus, recognition of dividing bacterial cells by the septin cytoskeleton is a powerful mechanism to restrict the proliferation of intracellular bacterial pathogens.
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Journal articleKarinou E, Schuster C, Pazos M, et al., 2018,
Inactivation of the monofunctional peptidoglycan glycosyltransferase SgtB allows Staphylococcus aureus to survive in the absence of lipoteichoic acid
, Journal of Bacteriology, Vol: 201, ISSN: 0021-9193The cell wall of Staphylococcus aureus is composed of peptidoglycan and the anionic polymers lipoteichoic acid (LTA) and wall teichoic acid. LTA is required for growth and normal cell morphology in S. aureus. Strains lacking LTA are usually viable only when grown under osmotically stabilizing conditions or after the acquisition of compensatory mutations. LTA-negative suppressor strains with inactivating mutations in gdpP, which resulted in increased intracellular c-di-AMP levels, were described previously. Here, we sought to identify factors other than c-di-AMP that allow S. aureus to survive without LTA. LTA-negative strains able to grow in unsupplemented medium were obtained and found to contain mutations in sgtB, mazE, clpX, or vraT. The growth improvement through mutations in mazE and sgtB was confirmed by complementation analysis. We also showed that an S. aureus sgtB transposon mutant, with the monofunctional peptidoglycan glycosyltransferase SgtB inactivated, displayed a 4-fold increase in the MIC of oxacillin, suggesting that alterations in the peptidoglycan structure could help bacteria compensate for the lack of LTA. Muropeptide analysis of peptidoglycans isolated from a wild-type strain and sgtB mutant strain did not reveal any sizable alterations in the peptidoglycan structure. In contrast, the peptidoglycan isolated from an LTA-negative ltaS mutant strain showed a significant reduction in the fraction of highly cross-linked peptidoglycan, which was partially rescued in the sgtB ltaS double mutant suppressor strain. Taken together, these data point toward an important function of LTA in cell wall integrity through its necessity for proper peptidoglycan assembly.
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Journal articlePissaridou P, Allsopp LP, Wettstadt S, et al., 2018,
The Pseudomonas aeruginosa T6SS-VgrG1b spike is topped by a PAAR protein eliciting DNA damage to bacterial competitors
, Proceedings of the National Academy of Sciences of the United States of America, Vol: 115, Pages: 12519-12524, ISSN: 0027-8424The type VI secretion system (T6SS) is a supramolecular complex involved in the delivery of potent toxins during bacterial competition. Pseudomonas aeruginosa possesses three T6SS gene clusters and several hcp and vgrG gene islands, the latter encoding the spike at the T6SS tip. The vgrG1b cluster encompasses seven genes whose organization and sequences are highly conserved in P. aeruginosa genomes, except for two genes that we called tse7 and tsi7. We show that Tse7 is a Tox-GHH2 domain nuclease which is distinct from other T6SS nucleases identified thus far. Expression of this toxin induces the SOS response, causes growth arrest and ultimately results in DNA degradation. The cytotoxic domain of Tse7 lies at its C terminus, while the N terminus is a predicted PAAR domain. We find that Tse7 sits on the tip of the VgrG1b spike and that specific residues at the PAAR–VgrG1b interface are essential for VgrG1b-dependent delivery of Tse7 into bacterial prey. We also show that the delivery of Tse7 is dependent on the H1-T6SS cluster, and injection of the nuclease into bacterial competitors is deployed for interbacterial competition. Tsi7, the cognate immunity protein, protects the producer from the deleterious effect of Tse7 through a direct protein–protein interaction so specific that toxin/immunity pairs are effective only if they originate from the same P. aeruginosa isolate. Overall, our study highlights the diversity of T6SS effectors, the exquisite fitting of toxins on the tip of the T6SS, and the specificity in Tsi7-dependent protection, suggesting a role in interstrain competition.
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Journal articleSanchez-Garrdio J, Sancho-Shimizu V, Shenoy A, 2018,
Regulated proteolysis of p62/SQSTM1 enables differential control of autophagy and nutrient sensing
, Science Signaling, Vol: 11, ISSN: 1937-9145The multidomain scaffold protein p62 (also called sequestosome-1) is involved in autophagy, antimicrobial immunity, and oncogenesis. Mutations in SQSTM1, which encodes p62, are linked to hereditary inflammatory conditions such as Paget’s disease of the bone, frontotemporal dementia (FTD), amyotrophic lateral sclerosis, and distal myopathy with rimmed vacuoles. Here, we report that p62 was proteolytically trimmed by the protease caspase-8 into a stable protein, which we called p62TRM. We found that p62TRM, but not full-length p62, was involved in nutrient sensing and homeostasis through the mechanistic target of rapamycin complex 1 (mTORC1). The kinase RIPK1 and caspase-8 controlled p62TRM production and thus promoted mTORC1 signaling. An FTD-linked p62 D329G polymorphism and a rare D329H variant could not be proteolyzed by caspase-8, and these noncleavable variants failed to activate mTORC1, thereby revealing the detrimental effect of these mutations. These findings on the role of p62TRM provide new insights into SQSTM1-linked diseases and mTORC1 signaling.
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Journal articleSwitzer A, Brown D, Wigneshweraraj S, 2018,
New insights into the adaptive transcriptional response to nitrogen starvation in Escherichia coli
, Biochemical Society Transactions, Vol: 46, Pages: 1721-1728, ISSN: 0300-5127Bacterial adaptive responses to biotic and abiotic stresses often involve large-scale reprogramming of the transcriptome. Since nitrogen is an essential component of the bacterial cell, the transcriptional basis of the adaptive response to nitrogen starvation has been well studied. The adaptive response to N starvation in Escherichia coli is primarily a ‘scavenging response’, which results in the transcription of genes required for the transport and catabolism of nitrogenous compounds. However, recent genome-scale studies have begun to uncover and expand some of the intricate regulatory complexities that underpin the adaptive transcriptional response to nitrogen starvation in E. coli. The purpose of this review is to highlight some of these new developments.
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