Biology subjects

Reder, A.

Publications and source records attributed to Reder, A..

5 recordsLinked to original sources

bioRxiv2025

Identification of novel human cellular substrates of Staphylococcus aureus serine protease SplB

Staphylococcus aureus colonizes up to one third of the human population yet retains the capacity to cause invasive, life-threatening infections. The growing prevalence of antimicrobial resistance further complicates treatment. A major contributor to the versatility of S. aureus is its broad repertoire of virulence factors, among which secreted proteases facilitate dissemination from colonization sites into deeper tissues. Twelve extracellular proteases are secreted, with the serine protease-like protein (Spl) family (SplA-SplF), encoded within a single operon, accounting for half of them. Despite this prominence, the pathophysiological roles and substrate specificities of the Spl proteases remain poorly understood. Here, we employed a direct protein-protein interaction approach to identify novel SplB substrates in human serum. We demonstrate that SplB cleaves three intermediate filament proteins, namely desmin, vimentin, and nestin, as well as heat shock protein {beta}1 and -enolase, which have not previously been recognized as targets of S. aureus proteases. Moreover, SplB was found to cleave native IgG, a feature otherwise described only for the glutamyl endopeptidase V8. These findings expand the host protein repertoire targeted by SplB and suggest broader roles for Spl proteases in immune evasion and tissue invasion.

immunology↗

bioRxiv2025

Unraveling proteomic chaos by independent component analysis - ClpX proficiency promotes the iron and oxygen limitation responses of Staphylococcus aureus and affects the intracellular bacterial behavior

In the opportunistic pathogen Staphylococcus aureus, protein homeostasis is largely mediated by the Caseinolytic protease (Clp) system. The proteases ClpXP and ClpCP are crucial for general and targeted proteolysis, which rely on the unfoldases ClpX and ClpC interacting with specific targets. However, the global effect on the proteome especially under infection-relevant stresses is not well-understood. To assess the effect of ClpX deficiency during infection-related processes, mass spectrometry-based global proteome profiles of S. aureus HG001 wild-type, an isogenic {Delta}clpX mutant, and a clpX complemented strain were recorded under control conditions as well as iron and oxygen limitation. The proteomic profiles revealed specific ClpX- and stress-dependent changes. A set of 24 robust stress-independent ClpX modulated proteins was identified and the stress-dependent influences were unraveled by independent component analysis (using the iModulon approach). These analyses revealed a role of ClpX in e.g., cell division, cell envelope homeostasis, the quinone stress response and prophage activation. Moreover, ClpX-dependent stress-specific effects were observed in the {Delta}clpX mutant, e.g. reduced induction of the heme uptake system under iron limitation and a dampened Rex-controlled oxygen limitation response. This revealed in particular that ClpX is central for heme homeostasis in S. aureus. Furthermore, in a Galleria infection model, the S. aureus {Delta}clpX mutant was attenuated compared to the wild-type HG001. This is consistent with a drastically reduced intracellular replication of the {Delta}clpX-mutant in cell culture-based infection experiments, however, high intracellular persistence of the {Delta}clpX mutant was also observed. This highlights the relevance of ClpX for bacterial fitness and virulence. ImportanceDuring infection processes, pathogens cope with host-mediated stressors. In response to those stressors, bacteria adapt their gene expression as well as their proteome profile. In the pathogen Staphylococcus aureus, protein homeostasis is mainly controlled by the Clp system. In particular, ClpX is the most conserved Clp unfoldase and is involved in overall regulation of virulence and bacterial fitness. However, the majority of ClpX targets remains elusive in S. aureus. With our proteomics approach and in depth data analysis, we provide a resource for global insight into ClpX-dependent adaptation of S. aureus physiology under infection-relevant conditions. Based on this, we uncover ClpXs role as a central player in the iron and oxygen limitation response. In addition, we demonstrate the importance of ClpX in S. aureus bacterial fitness in infection processes. However, reduced levels of ClpX lead to high intracellular persistence, which questions ClpXs suitability as a therapeutical target.

microbiology↗

bioRxiv2025

Requirement of ClpX for CtsR dissociation from its operator elements upon heat stress in Bacillus subtilis

A sudden increase in temperature triggers Bacillus subtilis to activate expression of stress-specific heat shock proteins of the CtsR (class three stress gene repressor) regulon to withstand the adverse conditions. Key members of this regulon, such as ATPases, proteolytic subunits and their adaptors, which can assemble to the functional Clp protease system, perform crucial roles in maintaining cellular proteostasis, while their transcription is repressed by CtsR during vegetative growth. Upon heat shock, a conformational change in a thermosensing glycine-rich loop causes CtsR to detach from its DNA operators, enabling the transcriptional activation of the regulon. Novel data from a clpX-deficient strain demonstrated that in addition, the presence of the ATPase ClpX is essential for the CtsR dissociation from its DNA binding site. To further elucidate this role of ClpX, we constructed a conditional clpX strain, in which clpX induction is decoupled from its native transcriptional control. This conditional expression system mimicked a clpX-deficient phenotype under non-inducing conditions and restored the wild-type phenotype upon induction. Our results indicate that the full induction of the CtsR regulon, particularly clpE, requires both heat and the presence of ClpX, thereby extending the current model for the transcriptional activation of genes repressed by CtsR.

microbiology↗

bioRxiv2025

Integrated genomic and proteomic analysis of the mouse-adapted Staphylococcus aureus strain JSNZ

Mouse-adapted Staphylococcus aureus strains have become increasingly relevant in infection research thanks to their ability to better recapitulate clinical infection dynamics in mouse models. However, detailed characterisations required to establish a corresponding reference strain are still lacking. The mouse-adapted CC88 strain JSNZ appears to be an ideal candidate for a reference strain, because CC88 is widespread among laboratory mice and frequently employed in mouse colonisation and infection models. Moreover, JSNZ demonstrates high genetic transformability comparable to that of commonly used laboratory strains. Here, we present a comprehensive genomic and proteomic characterisation of JSNZ. Whole genome sequencing was performed using a combination of short and long reads. Proteomic profiling was conducted under standard laboratory conditions in TSB and RPMI during exponential and stationary growth using LC-MS/MS. The updated, closed genome sequence of JSNZ was integrated into AureoWiki for user-friendly access and direct comparison to long-established reference strains. Genome data revealed a deletion in the restriction endonuclease gene hsdR, likely explaining the observed efficient transformation while retaining DNA modification capabilities. This positions JSNZ as a hub for genetic modification of other CC88 isolates. Proteomic profiling of JSNZ indicated broad similarity to common S. aureus reference strains. However, a striking exception was the novel serine protease Jep, which constituted approximately 75% of the exoproteome in stationary TSB cultures. Overall, these findings affirm JSNZ as a robust and genetically tractable model strain for murine S. aureus infection research and contribute a valuable standardised resource to enhance experimental reproducibility and cross-study consistency in the field. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/674026v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@130b864org.highwire.dtl.DTLVardef@199b1f2org.highwire.dtl.DTLVardef@171b6e0org.highwire.dtl.DTLVardef@9d371f_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Escherichia coli membrane microdomain SPFH protein HflC interacts with YajC and contributes to aminoglycoside and oxidative stress tolerance

Many eukaryotic membrane-dependent functions are often spatially and temporally regulated by membrane microdomains (FMMs) also known as lipid rafts. These domains are enriched in polyisoprenoid lipids and scaffolding proteins belonging to the Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) protein superfamily that was also identified in Gram-positive bacteria. By contrast, little is still known about FMMs in Gram-negative bacteria. In Escherichia coli K12, 4 SPFH proteins, YqiK, QmcA, HflK, and HflC, were shown to localize in discrete polar or lateral inner-membrane locations, raising the possibility that E. coli SPFH proteins could contribute to the assembly of inner-membrane FMMs and the regulation of cellular processes. Here we studied the determinant of the localization of QmcA and HflC and showed that FMM-associated cardiolipin lipid biosynthesis is required for their native localization pattern. Using Biolog phenotypic arrays, we showed that a mutant lacking all SPFH genes displayed increased sensitivity to aminoglycosides and oxidative stress that is due to the absence of HflKC. Our study therefore provides further insights into the contribution of SPFH proteins to stress tolerance in E. coli. IMPORTANCEEukaryotic cells often segregate physiological processes in cholesterol-rich functional membrane micro-domains. These domains are also called lipid rafts and contain proteins of the Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) superfamily, which are also present in prokaryotes but were mostly studied in Gram-positive bacteria. Here, we showed that the cell localization of the SPFH proteins QmcA and HflKC in the Gram-negative bacteria E. coli is altered in absence of cardiolipin lipid synthesis. This suggests that cardiolipins contribute to E. coli membrane microdomain assembly. Using a broad phenotypic analysis, we also showed that HflKC contribute to E. coli tolerance to aminoglycosides and oxidative stress. Our study, therefore, provides new insights into the cellular processes associated with SPFH proteins in E. coli.

microbiology↗