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Wolf-Dietrich Hardt

Publications and source records attributed to Wolf-Dietrich Hardt.

At least 19 recordsLinked to original sources
PubMed2025

The Gfr Uptake System Provides a Context-Dependent Fitness Advantage to Salmonella Typhimurium SL1344 During the Initial Gut Colonization Phase.

Salmonella enterica serovar Typhimurium (S. Tm) is a major cause of foodborne diarrhea. However, in healthy individuals, the microbiota typically restricts the growth of incoming pathogens, a protective mechanism termed colonization resistance (CR). To circumvent CR, Salmonella strains can utilize private nutrients that remain untapped by the resident microbiota. However, the metabolic pathways and environmental niches promoting pathogen growth are still not completely understood. Here, we investigate the significance of the gfr operon in gut colonization of S. Tm, which is essential for the utilization of fructoselysine (FL) and glucoselysine (GL). These Amadori compounds are present in heated foods with high protein and carbohydrate contents. We detected FL in both mouse chow and the intestinal tract of mice and showed that gfr mutants are attenuated during the initial phase of colonization in the murine model. Experiments in gnotobiotic mice and competition experiments with Escherichia coli suggest that gfr-dependent fitness advantage is context-dependent. We conclude that dietary Amadori products like FL can support S. Tm gut colonization, depending on the metabolic capacities of the microbiota.

Salmonella typhimurium↗
PubMed2025

Metabolic network reconstruction as a resource for analyzing Salmonella Typhimurium SL1344 growth in the mouse intestine.

Nontyphoidal Salmonella strains (NTS) are among the most common foodborne enteropathogens and constitute a major cause of global morbidity and mortality, imposing a substantial burden on global health. The increasing antibiotic resistance of NTS bacteria has attracted a lot of research on understanding their modus operandi during infection. Growth in the gut lumen is a critical phase of the NTS infection. This might offer opportunities for intervention. However, the metabolic richness of the gut lumen environment and the inherent complexity and robustness of the metabolism of NTS bacteria call for modeling approaches to guide research efforts. In this study, we reconstructed a thermodynamically constrained and context-specific genome-scale metabolic model (GEM) for S. Typhimurium SL1344, a model strain well-studied in infection research. We combined sequence annotation, optimization methods and in vitro and in vivo experimental data. We used GEM to explore the nutritional requirements, the growth limiting metabolic genes, and the metabolic pathway usage of NTS bacteria in a rich environment simulating the murine gut. This work provides insight and hypotheses on the biochemical capabilities and requirements of SL1344 beyond the knowledge acquired through conventional sequence annotation and can inform future research aimed at better understanding NTS metabolism and identifying potential targets for infection prevention.

Salmonella typhimurium↗
PubMed2024

Salmonella Typhimurium screen identifies shifts in mixed-acid fermentation during gut colonization.

How enteric pathogens adapt their metabolism to a dynamic gut environment is not yet fully understood. To investigate how Salmonella enterica Typhimurium (S.Tm) colonizes the gut, we conducted an in vivo transposon mutagenesis screen in a gnotobiotic mouse model. Our data implicate mixed-acid fermentation in efficient gut-luminal growth and energy conservation throughout infection. During initial growth, the pathogen utilizes acetate fermentation and fumarate respiration. After the onset of gut inflammation, hexoses appear to become limiting, as indicated by carbohydrate analytics and the increased need for gluconeogenesis. In response, S.Tm adapts by ramping up ethanol fermentation for redox balancing and supplying the TCA cycle with α-ketoglutarate for additional energy. Our findings illustrate how S.Tm flexibly adapts mixed fermentation and its use of the TCA cycle to thrive in the changing gut environment. Similar metabolic wiring in other pathogenic Enterobacteriaceae may suggest a broadly conserved mechanism for gut colonization.

Animals↗
PubMed2006

Salmonella type III secretion effectors: pulling the host cell's strings.

The enteric pathogen Salmonella employs type III secretion systems to transport a cocktail of effector proteins directly into its host cell. These effectors act in concert to control a variety of host cell processes to successfully invade intestinal cells and to establish an intracellular, replication-permissive niche. Recent studies reveal new insights into the molecular mechanisms that underlie effector protein injection, host cell invasion, and manipulation of vesicle trafficking induced by the interplay between multiple effectors and host systems. These findings corroborate the importance of spatio-temporal regulation of effector protein function for fine-tuned modulation of the host cell machinery.

Animals↗
PubMed2006

The chaperone binding domain of SopE inhibits transport via flagellar and SPI-1 TTSS in the absence of InvB.

Type III secretion systems (TTSS) are used by many Gram-negative pathogens for transporting effector proteins into eukaryotic host cells. Two modes of type III effector protein transport can be distinguished: transport into the surrounding medium (secretion) and cell-contact induced injection of effector proteins directly into the host cell cytosol (translocation). Two domains within the N-terminal regions of effector proteins determine the mode of transport. The amino terminal approximately 20 amino acids (N-terminal secretion signal, NSS) mediate secretion. In contrast, translocation generally requires the NSS, the adjacent approximately 100 amino acids (chaperone binding domain, CBD) and binding of the cognate chaperone to this CBD. TTSS are phylogenetically related to flagellar systems. Because both systems are expressed in Salmonella Typhimurium, correct effector protein transport involves at least two decisions: transport via the Salmonella pathogenicity island 1 (SPI-1) but not the flagellar TTSS (= specificity) and translocation into the host cell instead of secretion into the surrounding media (= transport mode). The mechanisms guiding these decisions are poorly understood. We have studied the S. Typhimurium effector protein SopE, which is specifically transported via the SPI-1 TTSS. Secretion and translocation strictly require the cognate chaperone InvB. Alanine replacement of amino acids 30-42 (and to some extent 44-54) abolished tight InvB binding, abolished translocation into the host cell and led to secretion of SopE via both, the flagellar and the SPI-1 TTSS. In clear contrast to wild-type SopE, secretion of SopE(Ala30-42) and SopE(Ala44-54) via the SPI-1 and the flagellar export system did not require InvB. These data reveal a novel function of the CBD: the CBD inhibits secretion of wild-type SopE via the flagellar and the SPI-1 TTSS in the absence of the chaperone InvB. Our data provide new insights into mechanisms ensuring specific effector protein transport by TTSS.

Amino Acid Sequence↗
PubMed2006

Chronic Salmonella enterica serovar Typhimurium-induced colitis and cholangitis in streptomycin-pretreated Nramp1+/+ mice.

Salmonella enterica subspecies 1 serovar Typhimurium is an enteric bacterial pathogen infecting a broad range of hosts. In susceptible Nramp1(-/-) (Slc11alpha1(-/-)) mice, serovar Typhimurium cannot efficiently colonize the intestine but causes a systemic typhoid-like infection. However, after pretreatment with streptomycin, these susceptible (C57BL/6 and BALB/c) mice develop acute serovar Typhimurium-induced colitis (M. Barthel et al., Infect. Immun. 71:2839-2858, 2003). It was not clear whether resistant Nramp1(+/+) (Slc11alpha1(+/+)) mouse strains would similarly develop colitis. Here we compared serovar Typhimurium infection in streptomycin-pretreated susceptible (C57BL/6) and resistant (DBA/2 and 129Sv/Ev) mouse strains: We found that acute colitis (days 1 and 3 postinfection) is strikingly similar in susceptible and resistant mice. In 129Sv/Ev mice we followed the serovar Typhimurium infection for as long as 6 weeks. After the initial phase of acute colitis, these animals developed chronic crypt-destructive colitis, including ulceration, crypt abscesses, pronounced mucosal and submucosal infiltrates, overshooting regeneration of the epithelium, and crypt branching. Moreover, we observed inflammation of the gall duct epithelium (cholangitis) in the 129Sv/Ev mice between days 14 and 43 of infection. Cholangitis was not attributable to side effects of the streptomycin treatment. Furthermore, chronic infection of 129Sv/Ev mice in a typhoid fever model did not lead to cholangitis. We propose that streptomycin-pretreated 129Sv/Ev mice provide a robust murine model for chronic enteric salmonellosis including complications such as cholangitis.

Animals↗
PubMed2006

Virulence of broad- and narrow-host-range Salmonella enterica serovars in the streptomycin-pretreated mouse model.

Salmonella enterica subspecies I serovars are common bacterial pathogens causing diseases ranging from enterocolitis to systemic infections. Some serovars are adapted to specific hosts, whereas others have a broad host range. The molecular mechanisms defining the virulence characteristics and the host range of a given S. enterica serovar are unknown. Streptomycin pretreated mice provide a surrogate host model for studying molecular aspects of the intestinal inflammation (colitis) caused by serovar Typhimurium (S. Hapfelmeier and W. D. Hardt, Trends Microbiol. 13:497-503, 2005). Here, we studied whether this animal model is also useful for studying other S. enterica subspecies I serovars. All three tested strains of the broad-host-range serovar Enteritidis (125109, 5496/98, and 832/99) caused pronounced colitis and systemic infection in streptomycin pretreated mice. Different levels of virulence were observed among three tested strains of the host-adapted serovar Dublin (SARB13, SD2229, and SD3246). Several strains of host restricted serovars were also studied. Two serovar Pullorum strains (X3543 and 449/87) caused intermediate levels of colitis. No intestinal inflammation was observed upon infection with three different serovar Paratyphi A strains (SARB42, 2804/96, and 5314/98) and one serovar Gallinarum strain (X3796). A second serovar Gallinarum strain (287/91) was highly virulent and caused severe colitis. This strain awaits future analysis. In conclusion, the streptomycin pretreated mouse model can provide an additional tool to study virulence factors (i.e., those involved in enteropathogenesis) of various S. enterica subspecies I serovars. Five of these strains (125109, 2229, 287/91, 449/87, and SARB42) are subject of Salmonella genome sequencing projects. The streptomycin pretreated mouse model may be useful for testing hypotheses derived from this genomic data.

Animals↗
PubMed2005

Real-time imaging of type III secretion: Salmonella SipA injection into host cells.

Many pathogenic and symbiotic Gram-negative bacteria employ type III secretion systems to inject "effector" proteins into eukaryotic host cells. These effectors manipulate signaling pathways to initiate symbiosis or disease. By using time-lapse microscopy, we have imaged delivery of the Salmonella type III effector protein SipA/SspA into animal cells in real time. SipA delivery mostly began 10-90 sec after docking and proceeded for 100-600 sec until the bacterial SipA pool (6 +/- 3 x 10(3) molecules) was exhausted. Similar observations were made for the effector protein SopE. This visualization of type III secretion in real time explains the efficiency of host cell manipulation by means of this virulence system.

Adhesins, Bacterial↗
PubMed2005

The Salmonella pathogenicity island (SPI)-2 and SPI-1 type III secretion systems allow Salmonella serovar typhimurium to trigger colitis via MyD88-dependent and MyD88-independent mechanisms.

Salmonella typhimurium can colonize the gut, invade intestinal tissues, and cause enterocolitis. In vitro studies suggest different mechanisms leading to mucosal inflammation, including 1) direct modulation of proinflammatory signaling by bacterial type III effector proteins and 2) disruption or penetration of the intestinal epithelium so that penetrating bacteria or bacterial products can trigger innate immunity (i.e., TLR signaling). We studied these mechanisms in vivo using streptomycin-pretreated wild-type and knockout mice including MyD88(-/-) animals lacking an adaptor molecule required for signaling via most TLRs. The Salmonella SPI-1 and the SPI-2 type III secretion systems (TTSS) contributed to inflammation. Mutants that retain only a functional SPI-1 (M556; sseD::aphT) or a SPI-2 TTSS (SB161; DeltainvG) caused attenuated colitis, which reflected distinct aspects of the colitis caused by wild-type S. typhimurium: M556 caused diffuse cecal inflammation that did not require MyD88 signaling. In contrast, SB161 induced focal mucosal inflammation requiring MyD88. M556 but not SB161 was found in intestinal epithelial cells. In the lamina propria, M556 and SB161 appeared to reside in different leukocyte cell populations as indicated by differential CD11c staining. Only the SPI-2-dependent inflammatory pathway required aroA-dependent intracellular growth. Thus, S. typhimurium can use two independent mechanisms to elicit colitis in vivo: SPI-1-dependent and MyD88-independent signaling to epithelial cells and SPI-2-dependent intracellular proliferation in the lamina propria triggering MyD88-dependent innate immune responses.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2005

Bacteriophage-encoded type III effectors in Salmonella enterica subspecies 1 serovar Typhimurium.

Salmonella spp. are Gram-negative bacteria which cause infections ranging from mild, self-limiting enterocolitis to systemic (typhoid) disease. Recent work has established that the genetic makeup varies considerably between different Salmonella strains. Phages play an important role in this diversity. In fact, Salmonella has emerged as a prime example for the involvement of virulence factor encoding phages in the emergence of new epidemic strains. Among other virulence factors, Salmonella enterica utilizes two specialized protein secretion systems termed type III secretion systems (TTSS) to deliver effector proteins into host cells which manipulate host cell signaling cascades. These two TTSS and several effectors are encoded within Salmonella pathogenicity islands 1 and 2. Some effectors including SopE, SspH1, SseI and SopE2 are encoded by phages or phage remnants. These phage-encoded effectors seem to be transferred between different Salmonella strains. They have attracted much interest because they might contribute to the evolution of Salmonella spp. Here we will focus on SopEPhi which encodes the SPI-1 effector SopE. It provides an excellent example to illustrate how horizontally transferred effector proteins are integrated into the complex regulatory network of a TTSS in a recipient bacterium. Additional data supporting the hypothesis are presented. This is a prerequisite to allow optimization of the bacterium host cell interaction by reassortment of the phage-encoded effector protein repertoire.

Bacterial Proteins↗
PubMed2005

A mouse model for S. typhimurium-induced enterocolitis.

Salmonella typhimurium has emerged as a model pathogen that manipulates host cells in a complex fashion, thus causing disease. In humans, S. typhimurium causes acute intestinal inflammation. Intriguingly, type III secreted virulence proteins have a central role in this process. At the cellular level, the functions of these factors are well characterized; at present, animal models are required for elucidating how these factors trigger inflammatory disease in vivo. Calf infection models have been employed successfully and, recently, a mouse model was identified: in streptomycin-pretreated mice, S. typhimurium causes acute colitis. This mouse model provides a new avenue for research into acute intestinal inflammation because it enables the manipulation and dissection of both the bacterial and host contributions to the disease in unsurpassed detail.

Animals↗
PubMed2005

Salmonella pathogenicity island 2-mediated overexpression of chimeric SspH2 proteins for simultaneous induction of antigen-specific CD4 and CD8 T cells.

Salmonella enterica serovar Typhimurium employs two different type III secretion systems (TTSS) encoded within Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) for targeting of effector proteins into the cytosol of eukaryotic cells during different stages of the infection cycle. The SPI1 TTSS translocates virulence factors across the plasma membrane when the bacterium initially contacts the host cell. In contrast, the SPI2 TTSS functions to translocate proteins across the membrane of the Salmonella-containing vacuole and promotes intracellular survival and replication. The aim of the present study was to directly compare the potentials of SPI1 and SPI2 type III effector proteins to act as carrier molecules for a heterologous antigen. The p60 protein of Listeria monocytogenes was used as a model antigen to construct chimeric SopE2 (SPI1), SifA (SPI2), and SspH2 (SPI2) proteins. SPI1- and SPI2-dependent up- and down-regulation of hybrid gene expression led to sequential translocation of p60 fusion proteins into the cytosol of Salmonella-infected macrophages. Mice orally immunized with recombinant Salmonella strains expressing these hybrid proteins revealed comparable numbers of p60-specific CD8 T cells. However, only overexpression of translocated SspH2/p60 from a medium-copy-number vector induced simultaneous antigen-specific CD4 and CD8 T-cell responses, suggesting that SspH2 is an attractive carrier molecule for foreign-protein delivery.

Amino Acid Sequence↗
PubMed2005

Comparison of Salmonella enterica serovar Typhimurium colitis in germfree mice and mice pretreated with streptomycin.

Salmonella enterica subspecies 1 serovar Typhimurium is a common cause of bacterial enterocolitis. Mice are generally protected from Salmonella serovar Typhimurium colonization and enterocolitis by their resident intestinal microflora. This phenomenon is called "colonization resistance" (CR). Two murine Salmonella serovar Typhimurium infection models are based on the neutralization of CR: (i) in specific-pathogen-free mice pretreated with streptomycin (StrSPF mice) antibiotics disrupt the intestinal microflora; and (ii) germfree (GF) mice are raised without any intestinal microflora, but their intestines show distinct physiologic and immunologic characteristics. It has been unclear whether the same pathogenetic mechanisms trigger Salmonella serovar Typhimurium colitis in GF and StrSPF mice. In this study, we compared the two colitis models. In both of the models Salmonella serovar Typhimurium efficiently colonized the large intestine and triggered cecum and colon inflammation starting 8 h postinfection. The type III secretion system encoded in Salmonella pathogenicity island 1 was essential in both disease models. Thus, Salmonella serovar Typhimurium colitis is triggered by similar pathogenetic mechanisms in StrSPF and GF mice. This is remarkable considering the distinct physiological properties of the GF mouse gut. One obvious difference was more pronounced damage and reduced regenerative response of the cecal epithelium in GF mice. Overall, StrSPF mice and GF mice provide similar but not identical models for Salmonella serovar Typhimurium colitis.

Animals↗
PubMed2004

Lack of mismatch correction facilitates genome evolution in mycobacteria.

In silico genome sequence analyses suggested that mycobacteria are devoid of the highly conserved mutLS-based post-replicative mismatch repair system. Here, we present the first biological evidence for the lack of a classical mismatch repair function in mycobacteria. We found that frameshifts, but not general mutation rates are unusually high in Mycobacterium smegmatis. However, despite the absence of mismatch correction, M. smegmatis establishes a strong barrier to recombination between homeologous DNA sequences. We show that 10-12% of DNA sequence heterology restricts initiation of recombination but not extension of heteroduplex DNA intermediates. Together, the lack of mismatch correction and a high stringency of initiation of homologous recombination provide an adequate strategy for mycobacterial genome evolution, which occurs by gene duplication and divergent evolution.

Base Pair Mismatch↗
PubMed2004

Role of the Salmonella pathogenicity island 1 effector proteins SipA, SopB, SopE, and SopE2 in Salmonella enterica subspecies 1 serovar Typhimurium colitis in streptomycin-pretreated mice.

Salmonella enterica subspecies 1 serovar Typhimurium (serovar Typhimurium) induces enterocolitis in humans and cattle. The mechanisms of enteric salmonellosis have been studied most extensively in calf infection models. The previous studies established that effector protein translocation into host cells via the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (TTSS) is of central importance in serovar Typhimurium enterocolitis. We recently found that orally streptomycin-pretreated mice provide an alternative model for serovar Typhimurium colitis. In this model the SPI-1 TTSS also plays a key role in the elicitation of intestinal inflammation. However, whether intestinal inflammation in calves and intestinal inflammation in streptomycin-pretreated mice are induced by the same SPI-1 effector proteins is still unclear. Therefore, we analyzed the role of the SPI-1 effector proteins SopB/SigD, SopE, SopE2, and SipA/SspA in elicitation of intestinal inflammation in the murine model. We found that sipA, sopE, and, to a lesser degree, sopE2 contribute to murine colitis, but we could not assign an inflammation phenotype to sopB. These findings are in line with previous studies performed with orally infected calves. Extending these observations, we demonstrated that in addition to SipA, SopE and SopE2 can induce intestinal inflammation independent of each other and in the absence of SopB. In conclusion, our data corroborate the finding that streptomycin-pretreated mice provide a useful model for studying the molecular mechanisms of serovar Typhimurium colitis and are an important starting point for analysis of the molecular events triggered by SopE, SopE2, and SipA in vivo.

Animals↗
PubMed2004

Flagella and chemotaxis are required for efficient induction of Salmonella enterica serovar Typhimurium colitis in streptomycin-pretreated mice.

Salmonella enterica subspecies 1 serovar Typhimurium is a common cause of gastrointestinal infections. The host's innate immune system and a complex set of Salmonella virulence factors are thought to contribute to enteric disease. The serovar Typhimurium virulence factors have been studied extensively by using tissue culture assays, and bovine infection models have been used to verify the role of these factors in enterocolitis. Streptomycin-pretreated mice provide an alternative animal model to study enteric salmonellosis. In this model, the Salmonella pathogenicity island 1 type III secretion system has a key virulence function. Nothing is known about the role of other virulence factors. We investigated the role of flagella in murine serovar Typhimurium colitis. A nonflagellated serovar Typhimurium mutant (fliGHI) efficiently colonized the intestine but caused little colitis during the early phase of infection (10 and 24 h postinfection). In competition assays with differentially labeled strains, the fliGHI mutant had a reduced capacity to get near the intestinal epithelium, as determined by fluorescence microscopy. A flagellated but nonchemotactic cheY mutant had the same virulence defects as the fliGHI mutant for causing colitis. In competitive infections, both mutants colonized the intestine of streptomycin-pretreated mice by day 1 postinfection but were outcompeted by the wild-type strain by day 3 postinfection. Together, these data demonstrate that flagella are required for efficient colonization and induction of colitis in streptomycin-pretreated mice. This effect is mostly attributable to chemotaxis. Recognition of flagellar subunits (i.e., flagellin) by innate immune receptors (i.e., Toll-like receptor 5) may be less important.

Animals↗
PubMed2004

Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion.

Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like "swarms" of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework.

Bacteria↗