Projects Molecular Microbiology

---

Research goals

The incidence of foodborne outbreaks caused by Enterobacteriaceae, including Salmonella enterica, remains substantial and constitutes a significant socioeconomic burden in Europe and worldwide. This is evidenced by the inclusion of Salmonella in the list of priority pathogens, which was recently published by the World Health Organization (WHO) to highlight urgent public health needs.

Colonization of the intestinal tract by S. Typhimurium. Route of infection and colonization of the small intestine (left). Electron micrographs of S. Typhimurium (right) [Schematic by Julia Horstmann, EM images by Manfred Rohde].

Salmonella enterica are motile, intracellular pathogens that employ multiple virulence factors, including flagella and needle-like injectisome devices, to efficiently colonize the host. The flagellum and injectisome are complex self-assembling nanomachines and their function relies on protein export via a conserved type-III secretion system. However, the molecular details of protein secretion via the type-III export apparatus remains largely obscure. In addition, complex regulatory mechanisms control the biosynthesis of the flagellum and virulence-related injectisome and substantial transcriptional cross talk between the various virulence factors exists that is poorly understood.

Our research concerning the molecular mechanisms of these fascinating nanomachines, in particular of the self-assembling bacterial flagellum, allows us to address fundamental research questions in microbiology and bacterial physiology, such as:

• 1. What are the molecular mechanisms of flagellum assembly and of the type-III protein secretion process?
• 2. What is the role of flagella and bacterial motility in the infection process?
• 3. How does Salmonella regulate its gene expression profile to adapt to diverse and hostile environments inside the host?

Elucidating the molecular mechanisms of the bacterial type-III protein secretion system and the life-style transition from the motile, planktonic state in the intestinal lumen to intracellular growth after invasion of the epithelium will be essential for a detailed understanding of the events required for the commitment of the bacteria to invasive diseases. Importantly, an in-depth knowledge of these mechanisms could be used to design rational strategies that counter the spread of gastrointestinal infections – urgently needed at a time when antibiotic resistance is increasing.

---

Molecular mechanisms of type-III protein secretion and self-assembly of the flagellum

The flagellum is a remarkably complex nanomachine. The flagellar filament is several times longer than the bacterial cell body and is made of thousands of building blocks of a single protein, flagellin. Flagellin is made inside the cell and secreted via a flagellum-specific type-III secretion system (T3SS) that uses energy derived from the proton motive force to pump it out of the cell. The exported flagellar building blocks travel then through a narrow channel within the flagellum and self-assemble at the tip.

However, it has been a mystery for several decades how bacteria manage to assemble the building blocks of flagella outside of the cell, where no discernible energy source is available. Recently, we used successive labelling of fragments of the flagellum with maleimide fluorochromes to determine the filament growth dynamics. Combining mathematical modeling, biochemical and single-cell fluorescent microscopy approaches, we could show that flagella grow through an injection-diffusion mechanism. Here, the growth rate of the flagellar filament (initial elongation speed of 100 nm/min) decreases with lenght of the flagellum (gradually to 20 nm/min) and thus explains why bacterial flagella do not continue to grow indefinitely.

In complementary projects, we study the protein export via T3SS, which is essential for assembly of the flagellum and virulence of many bacteria. While isolated components have been studied, purified T3SSs lack their integral membrane components, and thus mechanistic insights on substrate translocation, export apparatus assembly, and energy transfer to secretion is lacking.

In our research, we investigate the molecular function of the integral membrane components of the T3SS using a combination of bioinformatics, genetics and biochemical approaches. Genetic engineering allows us to elucidate inter and intra-molecular interactions of protein domains within the T3SS core complex. Various mutational approaches corroborate the biochemical analyses in context of secretion function and T3SS pore complex formation.

Fundamental open mechanistic questions in the field of type-III protein secretion are related to the mechanism of substrate translocation and coupling of the proton motive force to the export process, as well as to the nature of the type-III secretion signal, including the mechanism of substrate selectivity and how the T3SS discriminates between different substrate classes during the spatiotemporal assembly process of the flagellum. The mechanistic knowledge gained from the above-mentioned research program will form the basis for the development of novel anti-infectives that specifically target the bacterial type-III secretion system as virulence blockers.

Another research interest of the lab concerns the self-assembly of the bacterial flagellum. Here, we work on the long-standing question of how the bacterial flagellum controls the length of the extracellular hook structure that connects the basal body to the filament. In our work published in Molecular Microbiolology and The EMBO Journal, we showed that a molecular ruler protein is intermittently secreted throughout the assembly process and controls hook length in a stochastic process. The costs of motility are significant and thus, assembly of the flagellar structure is subject to strict control. We recently found that the molecular ruler mechanism controlling the length of the flagellar hook structure evolved in order to provide the optimal requirements for efficient motility.

---

Investigating the role of motility during pathogenesis

Many pathogenic bacteria including Salmonella are motile during a period of their life cycle. The ability to efficiently move in a directed manner confers distinct advantages. Potential benefits of motility in Salmonella include the ability to search for nutrients, to avoid toxic substances and facilitate contact with eukaryotic host cells for invasion. Consistently, the biosynthesis and assembly of substructures of the bacterial flagellum is tightly regulated and various control mechanisms are in place to determine stoichiometry and lengths of flagellar sub-assemblies, as well as flagella numbers. For instance, a molecular ruler mechanism evolved to control length of the flagellar hook, which is an essential, flexible joint that connects the basal body with the long, rigid filament.

The costs of motility are significant, including the metabolic burden of flagellar biosynthesis and the energetic expenses for flagellar rotation. Thus, it is not surprising that the flagellar structure is subject to strict control and presumably has evolved to provide the optimal requirements for efficient motility. While flagella and the ability to move is widely conserved in many pathogenic bacteria, the contribution and benefit of the chemotactic behavior during the infection process remains obscure. Many Salmonella serovars alternate expression of anti-genically distinct flagellin proteins, FliC and FljB. We recently analyzed the contribution of the flagellin phase to pathogenicity of Salmonella during the initial phase of the infection in the gastrointestinal lumen. We found that FljB-expressing bacteria were outcompeted by FliC-expressing bacteria during gastrointestinal colonization in the murine gastroenteritis infection model. Single-cell analysis of bacterial motility revealed differences in the run-and-tumble behavior during near-surface swimming, which explains the competitive advantage of FliC-expressing bacteria compared to FljB-expressing bacteria.

In addition, flagellin is post-translationally methylated in Salmonella. We recently found that methylation of the flagellar filament affects the interaction of the flagellated bacteria with hydrophobic host cell surfaces and thus has a pronounced effect on the pathogenicity of Salmonella.

Obtaining a mechanistic understanding of the motility behavior of pathogenic bacteria might unravel novel approaches for antimicrobial therapies. Interfering with motility processes has the potential to prevent infections from broad range of bacterial pathogens while having minimal impact on the commensal gut microbiota.

---

Regulatory mechanisms during gastrointestinal colonization

In order to succeed in invading its host, Salmonella employs a wide variety of virulence factors. These molecules are prime targets for the host’s immune system and in thus, the pathogen re-programs its gene expression profile from the motile, planktonic life style in the intestinal lumen to intracellular growth after invasion of the epithelium. However, a detailed understanding of how the pathogens achieve the correct spatiotemporal expression of their virulence determinants is lacking. Thus, a main research focus of our laboratory is to decipher the global gene regulatory networks that control spatio-temporal expression of the flagellum and connect the various virulence systems at different stages of the infection cycle.

We employ gene expression analyses in combination with single-cell, live-cell microscopy approaches to study the temporal gene expression dynamics during flagellar assembly and bacterial-epithelial cell interactions. In a complementary approach, we analyze the spatiotemporal gene expression dynamics during the infection process using RNA-sequencing of the in vivo transcriptome of Salmonella in the gastroenteritis mouse model. In collaboration with Till Strowig at the Helmholtz Centre for Infection Research, Braunschweig, we use gnotobiotic mice with defined microbioms to determine how the commensal microbiota contributes to colonization resistance against pathogenic Enterobacteriaceae.

This dual approach allows us to characterize the complex virulence-associated gene expression networks in the context of infection and address the gene expression re-programming of invading Salmonella in response to diverse microbiota environments.

---

Genetic engineering of Salmonella for pharmaceutical and biotechnological applications

Insights gained from our fundamental research on the regulation, function and structure of the flagellum and associated type-III secretion system allows us to translate our findings further into pharmaceutical and biotechnological applications.

In collaboration with Siegfried Weiß at the Helmholtz Centre for Infection Research, we investigate the immunogenic potential of the bacterial flagellum in an attempt to genetically engineer conditionally attenuated Salmonella bacteria for use in bacterium-mediated tumor therapy.

Cancer has become the second most frequent cause of death in industrialized countries. This and the drawbacks of routine therapies generate an urgent need for novel treatment options. Salmonella bacteria have an unique capability to specifically colonize tumor tissue and thus have the potential to be used in bacteria-mediated tumor therapy. However, applying appropriately modified Salmonella Typhimurium for therapy represents the major challenge of bacterium-mediated tumor therapy. In particular, the pathogenic Salmonella strains need to be attenuated for safe application in a tumor therapy application.

Towards this goal, we recently genetically engineered lipopolysaccharide (LPS) mutants of Salmonella and complemented the deletion mutants with the wild type copy of the respective gene expressed from an inducible arabinose promoter. This approach allowed for conditional attenuation and exhibited the best balance of attenuation and therapeutic benefit.

We further aim to isolate novel anti-infectives that specifically target the function of bacterial T3SS. Many pathogenic Enterobacteriaceae, including Salmonella, employ a wide variety of virulence factors - such as flagella, adhesins and secreted effectors by needle-like injectisome systems - to establish a productive infection and succeed in invading the host. In particular, the T3SS is conserved in both the bacterial flagellum and injectisome needle complex and is an essential virulence factor of many enterobacterial pathogens. Thus, the T3SS constitutes an excellent target for the development of novel broad-spectrum anti-virulence drugs.

---

Funding sources