Biology subjects

Michalik, S.

Publications and source records attributed to Michalik, S..

6 recordsLinked to original sources

bioRxiv2025

Immunoglobulin sub-class levels define inter-donor plasma variability: a longitudinal dual-lab study

Advancements in mass spectrometry have transformed plasma proteomics, allowing for high-throughput analysis of large cohorts. This study utilized the TIMES cohort, consisting of 51 healthy participants monitored monthly over 12 months, to evaluate intra- and inter-individual variability in the plasma proteome. Approximately 600 samples were analyzed in two independent laboratories, revealing strong correlations despite methodological differences. The study focused on the stability of the plasma proteome within donors over a year, with larger differences observed between donors. A support vector machine model achieved a 98% median classification accuracy, confirming stable, donor-specific proteomic profiles. Immunoglobulins, often overlooked in plasma proteomics, were found to contribute significantly to donor-specific signatures, showing pronounced inter-individual variability but remarkable intra-donor stability. In contrast, C-reactive protein and other inflammation markers exhibited significant temporal fluctuations and donor-specific baseline levels. This inter-laboratory study highlights the importance of longitudinal sampling for biomarker discovery, the robustness of MS-based proteomic workflows, and provides new insights into immunoglobulin dynamics and variability in human plasma. Synopsis O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/686385v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1e7b501org.highwire.dtl.DTLVardef@11c85d7org.highwire.dtl.DTLVardef@1971a1dorg.highwire.dtl.DTLVardef@1338061_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LILongitudinal analysis of individual plasma proteomes over a one-year period revealed low intra-individual variability albeit high inter-individual differences. C_LIO_LICross-site laboratory comparisons confirmed strong concordance in analytical performance and measurement consistency. C_LIO_LIConversion of relative protein intensities to absolute concentrations revealed immunoglobulin levels on par with established clinical reference ranges. C_LIO_LIDonors were distinguishable based on their unique plasma proteome. C_LIO_LIThe immunoglobulin sub-classes are key discriminators defining inter- and intra-donor variability C_LI

molecular biology↗

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↗

bioRxiv2025

Temperature-dependent regulation of bacterial cell division hydrolases by the coordinated action of a regulatory RNA and the ClpXP protease

A defining feature of bacteria is the peptidoglycan cell wall which provides structural integrity and prevents osmotic lysis. While peptidoglycan hydrolases are required for daughter cell separation, dysregulated cell wall degradation may result in cell lysis. The mechanisms allowing bacteria to control these deadly enzymes in response to environmental changes remain incompletely understood. Here, we find that in Staphylococcus aureus, temperature-dependent regulation of such hydrolases occurs by the coordinated action of a CHAP domain-specific regulatory RNA and the ClpXP protease. Using a proteomics approach, we identify a hitherto uncharacterized ClpXP controlled autolysin, CxcA, with a catalytic CHAP domain and show that it contributes to separation of daughter cells. CxcA is positively controlled by a non-coding RNA, named Rbc1 (for RNA binding to CHAP domain) transcribed from the antisense strand of cxcA. Notably, Rbc1 is capable of base pairing with RNAs encoding the CHAP domains of numerous cell wall hydrolases and we show that Rbc1 works in trans to upregulate the cell division hydrolase Sle1. Specifically, Rbc1 functions as a thermosensor allowing for upregulation of CxcA and Sle1 at low temperature where daughter cell separation is impeded. Interestingly, the Rbc1-mediated up-regulation of CxcA and Sle1 does not involve mRNA stabilization or increased translation; instead, Rbc1 depletion increases ClpXP-mediated degradation. In conclusion, we identify a novel cell division hydrolase that is highly conserved in Staphylococci and show that it is co-regulated with enzymes containing the catalytic CHAP domain via transcriptional regulation, an RNA-RNA temperature sensory mechanism and the ClpXP protease.

microbiology↗

bioRxiv2017

Natural Colonization of Laboratory Mice with Staphylococcus aureus Primes a Systemic Immune Response

BackgroundWhether mice are an appropriate model for S. aureus infection and vaccination studies is a matter of debate, because they are not considered as natural hosts of S. aureus. Sparked by an outbreak of S. aureus infections in laboratory mice, we investigated whether laboratory mice are commonly colonized with S. aureus and whether this might impact on infection experiments.\n\nMethodsWe characterized 99 S. aureus isolates from laboratory mice (spa typing, virulence gene PCR), and quantified murine antibodies using FlexMap technology.\n\nResultsSpecific-pathogen-free mice from various vendors were frequently colonized with S. aureus (0-21%). S. aureus was readily transmitted from murine parents to offspring, which became persistently colonized. Most murine isolates belonged to the lineage CC88 (54%). Murine strains showed features of host adaptation, such as absence of hlb-converting phages and superantigen genes, as well as enhanced coagulation of murine plasma. Importantly, S. aureus colonization induced a systemic IgG response specific for numerous S. aureus proteins, including several vaccine candidates.\n\nConclusionLaboratory mice are natural hosts of S. aureus and, therefore, provide better infection models than previously assumed. Pre-exposure to S. aureus is a possible confounder in S. aureus infection and vaccination studies.

immunology↗