7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Chair

Professor

Exploring Essential Genes in Staphylococcus aureus JE2-MRSA by: Transposition-Directed Insertion Site Sequencing (TraDIS)

Microbial Pathogenicity

Methicillin resistant Staphylococcus aureus (MRSA) is a major public health concerns worldwide causing a variety of clinical diseases including sepsis and abscess/skin infections. Transposon Directed Insertion-site Sequencing (TraDIS) is a genome-wide mutagenesis method, which allows simultaneous assaying of large transposon mutant libraries for exploring gene significance, functionality, and inter-genetic relationships in bacteria. In the current study, our goals were i) to construct a high-density Tn-mutants library in the JE2-MRSA strain of this species, and ii) and to identify the essential genes for in growth in vitro and for in vivo infection of skin and organs in a mouse model. We transducted the Tn-5 transposon/transposase complex into the strain by phage ø11 using an improved transduction protocol to create a high-density transposon library of 10^9 mutants. After the analysis by Bio-Tradis analysis pipeline, we found that the library containing 315,400 unique insertion sites (i.e. one insertion every 9 base pairs). We identified 213 essential genes with zero transposon insertion, suggesting that these genes were essential for growth on BHI agar medium, representing 8% of the total JE2 MRSA genome. Studies to identify essential genes for in vivo infection is ongoing and will be reported.

Oral Presentation

Prof. Dr.

Shiga Toxin Subunit Genes of Enterohemorrhagic Escherichia coli Are Not Transcribed in Equal Amounts

Introduction and Aim: The production of Shiga toxins is the major virulence factor of enterohemorrhagic Escherichia coli (EHEC). Shiga toxins are AB5 protein toxins consisting of a single enzymatically active A-subunit and a pentamer of non-covalently linked B-subunits. The genes encoding the Stx2 subunits, stxA2 and stxB2, are located in the late-regulated phage region within the genome of lambdoid prophages, downstream of the antiterminator gene Q and upstream of the genes encoding the phage lysis cassette. The aim of this study was to quantitatively analyze the relation of the transcription of stxA2 and stxB2 in five EHEC wildtype strains. Due to the operon structure of the stx genes, we hypothesized either a 1:1 transcription ratio, or due to the AB5 structure of the toxin, a 1:5 ratio.Material and Methods: To perform quantitative transcriptional analysis, total RNA was isolated and purified from E. coli O157:H7 strain HUSEC003, E. coli O157:H- strain HUSEC004, E. coli O103:H- strains HUSEC008, E. coli O26:H11 strain HUSEC018 and E. coli O104:H4 strain LB226692. Subsequently, qRT-PCR was performed to determine the transcript levels for stxA2, stxB2, and the housekeeping gene rrsB, which was used as an internal control.Results and Summary: Surprisingly, stxA2 was expressed approximately ~ 1.3 - 2.5 times stronger than stxB2 in all investigated EHEC wildtype strains. The analyzed genes were not expressed in the expected ratios, indicating that free A-subunits might circulate in the bacterial environment which do not find a B-pentamer for holotoxin formation. Further analysis on translational and protein level should help to clarify this phenomenon.

Improved purification of the recombinantly expressed A-subunit of Shiga toxin 2a with fast protein liquid chromatography

Introduction and Aim: Shiga Toxins are the major pathogenicity factors of enterohemorrhagic E. coli (EHEC). Stx are AB5 toxins, the A-subunit of which inhibits eukaryotic protein biosynthesis. The B-pentamer is responsible for binding to the receptor globotriaosylceramide. Since the A-subunit alone was already shown to be functional without its corresponding B-subunit, investigation and analysis of the mechanisms are of interest. Since mg amounts of toxin are needed for detailed analysis, current protocols of protein purification had to be optimized.The aim of the current study was to increase the yield of the pure A-subunit to a final yield of 200-300 µg StxA2a per litre expression culture.Materials and Methods: For the expression of StxA2a, the stxA2a gene was cloned into the vector pET45b(+) under the control of a T7 promotor, and transformed into E. coli BL21 (DE3) C43. In a first step the yield of StxA2a was optimized under different expression conditions (expression medium, expression time, expression temperature, IPTG concentrations). In a second step, StxA2a was purified with a single step purification using ion exchange chromatography.Results: The optimal expression of StxA2a was achieved in terrific broth after an incubation of 24 h at 30°C. The expression of StxA2a was induced with 1 mM IPTG. Since the His-tag present on the StxA2a was not accessible for purification on a metal-ion affinity column, ion exchange chromatography was used. Following this one step purification protocol, a yield of 567 µg pure StxA2a per litre expression culture was achieved.Summary: The optimization of the expression and purification of StxA2a leaded to a 5.5 times higher yield of pure StxA2a to former protocols. The purified StxA2a will be used for further studies on the cytotoxicity, the cellular uptake and the intracellular transport of StxA2a.

PD. Dr.

Flexibility of the PPI2 domain influences the activity of the periplasmic chaperone SurA in vivo

The periplasm of Gram-negative bacteria is comprised of a complex chaperone network, facilitating the transport of unfolded proteins to the outer membrane (OM). One major chaperone is SurA, which interacts with the β-barrel assembly machinery (BAM) complex and prevents aggregation of unfolded outer membrane proteins (OMPs) during their transport across the periplasm. SurA consists of three distinct domains: the NC-core and two peptidyl-prolyl-isomerase (PPI) domains PPI1 and PPI2. Previous studies revealed flexible &quot;closed to open&quot; conformational dynamics of these two PPI domains (1). While the binding of substrates involves mainly interactions within the PPI1/NC-core, the PPI2 domain can be linked to BAM-complex binding in vitro (2). Molecular dynamics simulations suggested, that the PPI2 domain forms salt bridges with the NC-core. Destabilization of these bonds via alanine substitutions results in increased SurA activity in vitro. Knowledge about PPI2 and the function of its interaction with the NC-core function remained scarce in vivo. Hence, we wanted to find out how interference with these interactions might impact the function of SurA in whole cells.To investigate the impact of weakened or stabilized interactions between PPI2 and the NC-core, we either created alanine substitutions or introduced disulfide bonds within the interaction surface and expressed the resulting SurA variants from the chromosome in an E. coli BW25113 ΔsurA strain. The activity of the SurA variants in vivo was then tested by probing OM permeability and the induction of a σE stress response using a PmicA-GFP reporter system. Thereby we aimed to quantify the cellular stress levels due to the accumulation of unfolded OMPs in the periplasm (3).Taken together, we found distinct phenotypes regarding OM integrity and σE stress in the periplasm of E. coli expressing SurA variants carrying changes in the PPI2/NC core interaction interface, that were correlated with the degree of flexibility of the PPI2 domain.Jia, M, et al. Biochemistry. 2020;59(35):3235-46Schiffrin, B, et al. Communications Biology. 2022;5(1):560Konovalova, A, et al. Proc Natl Acad Sci U S A. 2018;115(28):E6614-e21

Consequences of ClpX deficiency in Staphylococcus aureus under infection-mimicking conditions – a proteomic investigation

Introduction: Pathogenic bacteria have to cope with host-mediated stressors such as iron limitation and micro aerobic conditions. To do so, bacteria have to adapt gene expression as well as proteome profile and activity. In the pathogen S. aureus, protein homeostasis, and especially protein degradation and folding, is largely mediated by the Clp system consisting of the ClpP peptidase and the unfoldases ClpX and ClpC. The Clp protease system is crucial for general and targeted proteolysis, which relies on unfoldases interacting with specific targets. In particular, ClpX is the most conserved Clp unfoldase and it is involved in regulation of virulence. Goals: The majority of ClpX targets remains elusive in S. aureus despite the clear role of the unfoldases in virulence and fitness. We therefore aim to investigate the proteomic effect of ClpX deficiency under infection-mimicking conditions. Methods: For investigation of the effect of ClpX deficiency the S. aureus mutant strain HG001 ∆clpX::km and the complemented strain HG001 ∆clpX::km pTripleTREP_clpX were constructed employing the newly developed plasmid systems pSauSE (allele exchange plasmid) and pTripleTREP (controllable expression plasmid). Mass spectrometry-based proteome profiles were then recorded for S. aureus HG001 and the constructed isogenic mutants under aerobic iron-rich control conditions, micro aerobic conditions and iron depleted conditions in exponential and stationary growth phase. Results: The proteomic profiles reveal ClpX- and condition-dependent changes. Known ClpX-targets such as Spx, Sle1 and Spa were confirmed. Under infection-relevant conditions, iron limitation generally induced the Fur regulon and repressed the IsrR targetome, and under micro aerobic condition the NreBC regulon and the Rex regulon are induced, as expected. Moreover, ClpX-dependent condition-specific effects, such as a sub regulon of the known Fur regulon which is less responsive to the provoked iron limitation in the ∆clpX mutant, were also observed. Summary: By presenting our proteomic study, we provide a global insight into ClpX-dependent adaptation of S. aureus physiology under infection-relevant conditions.

Plasmodium falciparum utilizes proteins of the complement and fibrinolysis pathway for red blood cell invasion

Eukaryotic Pathogens

The intracellular parasite Plasmodium falciparum causes malaria tropica, the severest form of malaria in the WHO African region. With over 619,000 deaths in 2022, malaria is still one of the deadliest diseases worldwide. The symptomatic phase includes fevers, head- and body aches, and diarrhea. In erythrocytes, the parasite develops via the ring, then trophozoite stages into mature schizonts, which form up to 32 daughter merozoites (MZs) that are released and invade new red blood cells (RBCs). In the bloodstream, the extracellular MZs are susceptible to the human complement system. To avoid complement-mediated lysis, the parasites bind to the complement inhibitor factor H (FH) or to plasminogen, a protein in the fibrinolysis system. There is currently little information on how these proteins influence MZ invasion of RBCs. Therefore, we performed MZ invasion assays to investigate how proteins of the FH family, plasminogen, and other key alternative pathway proteins influence their entry into RBCs. We found that in the presence of C3b but also under FHR1-depleted conditions, the numbers of intraerythrocytic parasites increased. Furthermore, under plasminogen-deficient conditions, RBC infection by MZs was reduced, while the addition of plasminogen led to increased invasion rates independent of active complement. In conclusion, we hypothesize that MZs utilize proteins of the complement and the fibrinolysis system to facilitate erythrocyte attachment for efficient and successful invasion and intraerythrocytic replication.

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