Ex vivo infection model for Francisella using human lung tissue.

Introduction: Tularemia is mainly caused by Francisella tularensis (Ft) subsp. tularensis (Ftt) and Ft subsp. holarctica (Ftt) in humans and in more than 200 animal species including rabbits and hares. Human clinical manifestations depend on the route of infection and range from flu-like symptoms to severe pneumonia with a mortality rate up to 60% without treatment. So far, only 2D cell culture and animal models are used to study Francisella virulence, but the gained results are transferable to human infections only to a certain extent. Method: In this study, we firstly established an ex vivo human lung tissue infection model using different Francisella strains: Ftt Life Vaccine Strain (LVS), Ftt LVS ΔiglC, Ftt human clinical isolate A-660 and a German environmental Francisella species strain W12-1067 (F-W12). Human lung tissue was used to determine the colony forming units and to detect infected cell types by using spectral immunofluorescence and electron microscopy. Chemokine and cytokine levels were measured in culture supernatants. Results: Only LVS and A-660 were able to grow within the human lung explants, whereas LVS ΔiglC and F-W12 did not replicate. Using human lung tissue, we observed a greater increase of bacterial load per explant for patient isolate A-660 compared to LVS, whereas a similar replication of both strains was observed in cell culture models with human macrophages. Alveolar macrophages were mainly infected in human lung tissue, but Ftt was also sporadically detected within white blood cells. Although Ftt replicated within lung tissue, an overall low induction of pro-inflammatory cytokines and chemokines was observed. A-660-infected lung explants secreted slightly less of IL-1ß, MCP-1, IP-10 and IL-6 compared to Ftt LVS-infected explants, suggesting a more repressed immune response for patient isolate A-660. When LVS and A-660 were used for simultaneous co-infections, only the ex vivo model reflected the less virulent phenotype of LVS, as it was outcompeted by A-660. Conclusion: We successfully implemented an ex vivo infection model using human lung tissue for Francisella. The model delivers considerable advantages and is able to discriminate virulent Francisella from less- or non-virulent strains and can be used to investigate the role of specific virulence factors.

Phenotypic and genotypic discrimination of Francisella tularensis ssp. holarctica clades.

Francisella tularensis is the causative agent of tularemia, a zoonotic disease with a wide host range. F. tularensis ssp. holarctica (Fth) is of clinical relevance for European countries, including Germany. Whole genome sequencing methods, including canonical Single Nucleotide Polymorphism (canSNP) typing and whole genome SNP typing, have revealed that European Fth strains belong to a few monophyletic populations. The majority of German Fth isolates belong to two basal phylogenetic clades B.6 (biovar I) and B.12 (biovar II). Strains of B.6 and B.12 seem to differ in their pathogenicity, and it has been shown that strains of biovar II are resistant against erythromycin. In this study, we present data corroborating our previous data demonstrating that basal clade B.12 can be divided into clades B.71 and B.72. By applying phylogenetic whole genome analysis as well as proteome analysis, we could verify that strains of these two clades are distinct from one another. This was confirmed by measuring the intensity of backscatter light on bacteria grown in liquid media. Strains belonging to clades B.6, B.71 or B.72 showed clade-specific backscatter growth curves. Furthermore, we present the whole genome sequence of strain A-1341, as a reference genome of clade B.71, and whole proteomes comparison of Fth strains belonging to clades B.6, B.71 and B.72. Further research is necessary to investigate phenotypes and putative differences in pathogenicity of the investigated different clades of Fth to better understand the relationship between observed phenotypes, pathogenicity and distribution of Fth strains.

Ulceroglandular form of tularemia after squirrel bite: a case report.

BACKGROUND: The diagnosis of tularemia is not often considered in Germany as the disease is still rare in this country. Nonetheless, Francisella tularensis, the causative agent of tularemia, can infect numerous animal species and should, therefore, not be neglected as a dangerous pathogen. Tularemia can lead to massively swollen lymph nodes and might even be fatal without antibiotic treatment. To our knowledge, the case described here is the first report of the disease caused by a squirrel bite in Germany. CASE PRESENTATION: A 59-year-old German woman with a past medical history of hypothyroidism and cutaneous lupus erythematosus presented at the emergency room at St. Katharinen Hospital with ongoing symptoms and a swollen right elbow persisting despite antibiotic therapy with cefuroxime for 7 days after she had been bitten (right hand) by a wild squirrel (Eurasian red squirrel). After another 7 days of therapy with piperacillin/tazobactam, laboratory analysis using real-time polymerase chain reaction (PCR) confirmed the suspected diagnosis of tularemia on day 14. After starting the recommended antibiotic treatment with ciprofloxacin, the patient recovered rapidly. CONCLUSION: This is the first report of a case of tularemia caused by a squirrel bite in Germany. A naturally infected squirrel has recently been reported in Switzerland for the first time. The number of human cases of tularemia has been increasing over the last years and, therefore, tularemia should be taken into consideration as a diagnosis, especially in a patient bitten by an animal who also presents with headache, increasing pain, lymphadenitis, and fever, as well as impaired wound healing. The pathogen can easily be identified by a specific real-time PCR assay of wound swabs and/or by antibody detection, for example by enzyme-linked immunosorbent assay (ELISA), if the incident dates back longer than 2 weeks.

Metabolic adaption of Legionella pneumophila during intracellular growth in Acanthamoeba castellanii.

The metabolism of Legionella pneumophila strain Paris was elucidated during different time intervals of growth within its natural host Acanthamoeba castellanii. For this purpose, the amoebae were supplied after bacterial infection (t =0 h) with 11 mM [U-13C6]glucose or 3 mM [U-13C3]serine, respectively, during 0-17 h, 17-25 h, or 25-27 h of incubation. At the end of these time intervals, bacterial and amoebal fractions were separated. Each of these fractions was hydrolyzed under acidic conditions. 13C-Enrichments and isotopologue distributions of resulting amino acids and 3-hydroxybutyrate were determined by gas chromatography - mass spectrometry. Comparative analysis of the labelling patterns revealed the substrate preferences, metabolic pathways, and relative carbon fluxes of the intracellular bacteria and their amoebal host during the time course of the infection cycle. Generally, the bacterial infection increased the usage of exogenous glucose via glycolysis by A. castellanii. In contrast, carbon fluxes via the amoebal citrate cycle were not affected. During the whole infection cycle, intracellular L. pneumophila incorporated amino acids from their host into the bacterial proteins. However, partial bacterial de novo biosynthesis from exogenous 13C-Ser and, at minor rates, from 13C-glucose could be shown for bacterial Ala, Asp, Glu, and Gly. More specifically, the catabolic usage of Ser increased during the post-exponential phase of intracellular growth, whereas glucose was utilized by the bacteria throughout the infection cycle and not only late during infection as assumed on the basis of earlier in vitro experiments. The early usage of 13C-glucose by the intracellular bacteria suggests that glucose availability could serve as a trigger for replication of L. pneumophila inside the vacuoles of host cells.

First Description of a Temperate Bacteriophage (vB_FhiM_KIRK) of Francisella hispaniensis Strain 3523.

Here we present the characterization of a Francisella bacteriophage (vB_FhiM_KIRK) including the morphology, the genome sequence and the induction of the prophage. The prophage sequence (FhaGI-1) has previously been identified in F. hispaniensis strain 3523. UV radiation induced the prophage to assemble phage particles consisting of an icosahedral head (~52 nm in diameter), a tail of up to 97 nm in length and a mean width of 9 nm. The double stranded genome of vB_FhiM_KIRK contains 51 open reading frames and is 34,259 bp in length. The genotypic and phylogenetic analysis indicated that this phage seems to belong to the Myoviridae family of bacteriophages. Under the conditions tested here, host cell (Francisella hispaniensis 3523) lysis activity of KIRK was very low, and the phage particles seem to be defective for infecting new bacterial cells. Nevertheless, recombinant KIRK DNA was able to integrate site-specifically into the genome of different Francisella species after DNA transformation.

Complete Genome Sequence of Francisella tularensis subsp. holarctica Strain A271_1 (FDC408), Isolated from a Eurasian Beaver (Castor fiber).

Here, we report the complete genome sequence of Francisella tularensis subsp. holarctica strain A271_1, isolated from a Eurasian beaver (Castor fiber) in 2012 in the Berlin/Brandenburg region, Germany.

Outbreak of Tularemia in a Group of Hunters in Germany in 2018-Kinetics of Antibody and Cytokine Responses.

In November 2018, an outbreak of tularemia occurred among hare hunters in Bavaria, Germany. At least one infected hare was confirmed as the source of infection. A number of hunting dogs showed elevated antibody titers to Francisella tularensis, but the absence of titer increases in subsequent samples did not point to acute infections in dogs. Altogether, 12 persons associated with this hare hunt could be diagnosed with acute tularemia by detection of specific antibodies. In nine patients, the antibody and cytokine responses could be monitored over time. Eight out of these nine patients had developed detectable antibodies three weeks after exposure; in one individual the antibody response was delayed. All patients showed an increase in various cytokines and chemokines with a peak for most mediators in the first week after exposure. Cytokine levels showed individual variations, with high and low responders. The kinetics of seroconversion has implications on serological diagnoses of tularemia.

Francisella tularensis Subspecies holarctica and Tularemia in Germany.

Tularemia is a zoonotic disease caused by Francisella tularensis a small, pleomorphic, facultative intracellular bacterium. In Europe, infections in animals and humans are caused mainly by Francisella tularensis subspecies holarctica. Humans can be exposed to the pathogen directly and indirectly through contact with sick animals, carcasses, mosquitoes and ticks, environmental sources such as contaminated water or soil, and food. So far, F. tularensis subsp. holarctica is the only Francisella species known to cause tularemia in Germany. On the basis of surveillance data, outbreak investigations, and literature, we review herein the epidemiological situation-noteworthy clinical cases next to genetic diversity of F. tularensis subsp. holarctica strains isolated from patients. In the last 15 years, the yearly number of notified cases of tularemia has increased steadily in Germany, suggesting that the disease is re-emerging. By sequencing F. tularensis subsp. holarctica genomes, knowledge has been added to recent findings, completing the picture of genotypic diversity and geographical segregation of Francisella clades in Germany. Here, we also shortly summarize the current knowledge about a new Francisella species (Francisella sp. strain W12-1067) that has been recently identified in Germany. This species is the second Francisella species discovered in Germany.

Myo-Inositol as a carbon substrate in Francisella and insights into the metabolism of Francisella sp. strain W12-1067.

Recently, a new environmental Francisella strain, Francisella sp. strain W12-1067, has been identified in Germany. This strain is negative for the Francisella pathogenicity island (FPI) but exhibits a putative alternative type VI secretion system. Some known virulence factors of Francisella are present, but the pathogenic capacity of this species is not known yet. In silico genome analysis reveals the presence of a gene cluster tentatively enabling myo-inositol (MI) utilization via a putative inositol oxygenase. Labelling experiments starting from 2H-inositol demonstrate that this gene cluster is indeed involved in the metabolism of MI. We further show that, under in vitro conditions, supply of MI increases growth rates of strain W12-1067 in the absence of glucose and that the metabolism of MI is strongly reduced in a W12-1067 mutant lacking the MI gene cluster. The positive growth effect of MI in the absence of glucose is restored in this mutant strain by introducing the complete MI gene cluster. F. novicida Fx1 is also positive for the MI metabolizing gene cluster and MI again increases growth in a glucose-free medium, in contrast to F. novicida strain U112, which is shown to be a natural mutant of the MI metabolizing gene cluster. Labelling experiments of Francisella sp. strain W12-1067 in medium T containing 13C-glucose, 13C-serine or 13C-glycerol as tracers suggest a bipartite metabolism where glucose is mainly metabolized through glycolysis, but not through the Entner-Doudoroff pathway or the pentose phosphate pathway. Carbon flux from 13C-glycerol and 13C-serine is less active, and label from these tracers is transferred mostly into amino acids, lactate and fatty acids. Together, the metabolism of Francisella sp. strain W12-1067 seems to be more related to the respective one in F. novicida rather than in F. tularensis subsp. holarctica.

Genetic Diversity and Spatial Segregation of Francisella tularensis Subspecies holarctica in Germany.

Francisella tularensis is an intracellular pleomorphic bacterium and the causative agent of tularemia, a zoonotic disease with a wide host range. Among the F. tularensis subspecies, especially F. tularensis subsp. holarctica is of clinical relevance for European countries. The study presented herein focuses namely on genetic diversity and spatial segregation of F. tularensis subsp. holarctica in Germany, as still limited information is available. The investigation is based on the analysis of 34 F. tularensis subsp. holarctica isolates and one draft genome from an outbreak strain. The isolates were cultured from sample material being that of primarily human patients (n = 25) and free-living animals (n = 9). For six of 25 human isolates, epidemiological links between disease onset and tick bites could be established, confirming the importance of arthropod linked transmission of tularemia in Germany. The strains were assigned to three of four major F. tularensis subsp. holarctica clades: B.4, B.6, and B.12. Thereby, B.6 and B.12 clade members were predominantly found; only one human isolate was assigned to clade B.4. Also, it turned out that eight isolates which caused pneumonia in patients clustered into the B.6 clade. Altogether, eight different final subclades were assigned to clade B.6 (biovar I, erythromycin sensitive) and six to B.12 (biovar II, erythromycin resistant) in addition to one new final B.12 subclade. Moreover, for 13 human and 3 animal isolates, final subclade subdivisions were not assigned (B.12 subdivisions B.33 and B.34, and B.6 subdivision B.45) because official nomenclatures are not available yet. This gives credit to the genetic variability of F. tularensis subsp. holarctica strains in Germany. The results clearly point out that the given genetic diversity in Germany seems to be comparably high to that found in other European countries including Scandinavian regions. A spatial segregation of B.6 and B.12 strains was found and statistically confirmed, and B.12 clade members were predominantly found in eastern parts and B.6 members more in western to southern parts of Germany. The portion of B.12 clade members in northeastern parts of Germany was 78.5% and in southwestern parts 1.9%.

Screen for fitness and virulence factors of Francisella sp. strain W12-1067 using amoebae.

Francisella tularensis is the causative agent of the human disease referred to as tularemia. Other Francisella species are known but less is understood about their virulence factors. The role of environmental amoebae in the life-cycle of Francisella is still under discussion. Francisella sp. strain W12-1067 (F-W12) is an environmental Francisella isolate recently identified in Germany which is negative for the Francisella pathogenicity island, but exhibits a putative alternative type VI secretion system. Putative virulence factors have been identified in silico in the genome of F-W12. In this work, we established a "scatter screen", used earlier for pathogenic Legionella, to verify experimentally and identify candidate fitness factors using a transposon mutant bank of F-W12 and Acanthamoeba lenticulata as host organism. In these experiments, we identified 79 scatter clones (amoeba sensitive), which were further analyzed by an infection assay identifying 9 known virulence factors, but also candidate fitness factors of F-W12 not yet described as fitness factors in Francisella. The majority of the identified genes encoded proteins involved in the synthesis or maintenance of the cell envelope (LPS, outer membrane, capsule) or in the metabolism (glycolysis, gluconeogenesis, pentose phosphate pathway). Further 13C-flux analysis of the Tn5 glucokinase mutant strain revealed that the identified gene indeed encodes the sole active glucokinase in F-W12. In conclusion, candidate fitness factors of the new Francisella species F-W12 were identified using the scatter screen method which might also be usable for other Francisella species.

Molecular identification of the source of an uncommon tularaemia outbreak, Germany, autumn 2016.

BackgroundIn 2016, an uncommon outbreak of oropharyngeal tularaemia involving six human cases occurred in Germany, caused by drinking contaminated fresh must after a grape harvest.AimWe describe the details of laboratory investigations leading to identification of the outbreak strain, its characterisation by next generation sequencing (NGS) and the finding of the possible source of contamination.MethodsWe incubated wine samples in different media and on agar plates. NGS was performed on DNA isolated from young wine, sweet reserve and an outbreak case's lymph node. A draft genome of the outbreak strain was generated. Vertebrate-specific PCRs using primers targeting the mitochondrial cytochrome b gene and product analyses by blast search were used to identify the putative source of must contamination.ResultsNo bacterial isolate could be obtained. Analysis of the draft genome sequence obtained from the sweet reserve attributed this sequence to Francisella tularensis subsp. holarctica, belonging to the B.12/B.34 phylogenetic clade (erythromycin-resistant biovar II). In addition, the DNA sequence obtained from the case's isolate supported our hypothesis that infection was caused by drinking contaminated must. The vertebrate-specific cytochrome b sequence derived from the young wine and the sweet reserve could be assigned to Apodemus sylvaticus (wood mouse), suggesting that a wood mouse infected with F. tularensis may have contaminated the must.ConclusionThe discovered source of infection and the transmission scenario of F. tularensis in this outbreak have not been observed previously and suggest the need for additional hygienic precautionary measures when processing and consuming freshly pressed must.

The Pathometabolism of Legionella Studied by Isotopologue Profiling.

Metabolic pathways and fluxes can be analyzed under in vivo conditions by incorporation experiments using general 13C-labelled precursors. On the basis of the isotopologue compositions in amino acids or other metabolites, the incorporation rates of the supplied precursors and the pathways of their utilization can be studied in considerable detail. In this chapter, the method of isotopologue profiling is illustrated with recent work on the metabolism of intracellular living Legionella pneumophila.

Tularemia in Germany-A Re-emerging Zoonosis.

Tularemia, also known as "rabbit fever," is a zoonosis caused by the facultative intracellular, gram-negative bacterium Francisella tularensis. Infection occurs through contact with infected animals (often hares), arthropod vectors (such as ticks or deer flies), inhalation of contaminated dust or through contaminated food and water. In this review, we would like to provide an overview of the current epidemiological situation in Germany using published studies and case reports, an analysis of recent surveillance data and our own experience from the laboratory diagnostics, and investigation of cases. While in Germany tularemia is a rarely reported disease, there is evidence of recent re-emergence. We also describe some peculiarities that were observed in Germany, such as a broad genetic diversity, and a recently discovered new genus of Francisella and protracted or severe clinical courses of infections with the subspecies holarctica. Because tularemia is a zoonosis, we also touch upon the situation in the animal reservoir and one-health aspects of this disease. Apparently, many pieces of the puzzle need to be found and put into place before the complex interaction between wildlife, the environment and humans are fully understood. Funding for investigations into rare diseases is scarce. Therefore, combining efforts in several countries in the framework of international projects may be necessary to advance further our understanding of this serious but also scientifically interesting disease.

Construction of a New Phage Integration Vector pFIV-Val for Use in Different Francisella Species.

We recently identified and described a putative prophage on the genomic island FhaGI-1 located within the genome of Francisella hispaniensis AS02-814 (F. tularensis subsp. novicida-like 3523). In this study, we constructed two variants of a Francisella phage integration vector, called pFIV1-Val and pFIV2-Val (Francisella Integration Vector-tRNAVal-specific), using the attL/R-sites and the site-specific integrase (FN3523_1033) of FhaGI-1, a chloramphenicol resistance cassette and a sacB gene for counter selection of transformants against the vector backbone. We inserted the respective sites and genes into vector pUC57-Kana to allow for propagation in Escherichia coli. The constructs generated a circular episomal form in E. coli which could be used to transform Francisella spp. where FIV-Val stably integrated site specifically into the tRNAVal gene of the genome, whereas pUC57-Kana is lost due to counter selection. Functionality of the new vector was demonstrated by the successfully complementation of a Francisella mutant strain. The vectors were stable in vitro and during host-cell infection without selective pressure. Thus, the vectors can be applied as a further genetic tool in Francisella research, expanding the present genetic tools by an integrative element. This new element is suitable to perform long-term experiments with different Francisella species.

The Flagellar Regulon of Legionella-A Review.

The Legionella genus comprises more than 60 species. In particular, Legionella pneumophila is known to cause severe illnesses in humans. Legionellaceae are ubiquitous inhabitants of aquatic environments. Some Legionellaceae are motile and their motility is important to move around in habitats. Motility can be considered as a potential virulence factor as already shown for various human pathogens. The genes of the flagellar system, regulator and structural genes, are structured in hierarchical levels described as the flagellar regulon. Their expression is modulated by various environmental factors. For L. pneumophila it was shown that the expression of genes of the flagellar regulon is modulated by the actual growth phase and temperature. Especially, flagellated Legionella are known to express genes during the transmissive phase of growth that are involved in the expression of virulence traits. It has been demonstrated that the alternative sigma-28 factor is part of the link between virulence expression and motility. In the following review, the structure of the flagellar regulon of L. pneumophila is discussed and compared to other flagellar systems of different Legionella species. Recently, it has been described that Legionella micdadei and Legionella fallonii contain a second putative partial flagellar system. Hence, the report will focus on flagellated and non-flagellated Legionella strains, phylogenetic relationships, the role and function of the alternative sigma factor (FliA) and its anti-sigma-28 factor (FlgM).

Differential Substrate Usage and Metabolic Fluxes in Francisella tularensis Subspecies holarctica and Francisella novicida.

Francisella tularensis is an intracellular pathogen for many animals causing the infectious disease, tularemia. Whereas F. tularensis subsp. holarctica is highly pathogenic for humans, F. novicida is almost avirulent for humans, but virulent for mice. In order to compare metabolic fluxes between these strains, we performed 13C-labeling experiments with F. tularensis subsp. holarctica wild type (beaver isolate), F. tularensis subsp. holarctica strain LVS, or F. novicida strain U112 in complex media containing either [U-13C6]glucose, [1,2-13C2]glucose, [U-13C3]serine, or [U-13C3]glycerol. GC/MS-based isotopolog profiling of amino acids, polysaccharide-derived glucose, free fructose, amino sugars derived from the cell wall, fatty acids, 3-hydroxybutyrate, lactate, succinate and malate revealed uptake and metabolic usage of all tracers under the experimental conditions with glucose being the major carbon source for all strains under study. The labeling patterns of the F. tularensis subsp. holarctica wild type were highly similar to those of the LVS strain, but showed remarkable differences to the labeling profiles of the metabolites from the F. novicida strain. Glucose was directly used for polysaccharide and cell wall biosynthesis with higher rates in F. tularensis subsp. holarctica or metabolized, with higher rates in F. novicida, via glycolysis and the non-oxidative pentose phosphate pathway (PPP). Catabolic turnover of glucose via gluconeogenesis was also observed. In all strains, Ala was mainly synthesized from pyruvate, although no pathway from pyruvate to Ala is annotated in the genomes of F. tularensis and F. novicida. Glycerol efficiently served as a gluconeogenetic substrate in F. novicida, but only less in the F. tularensis subsp. holarctica strains. In any of the studied strains, serine did not serve as a major substrate and was not significantly used for gluconeogenesis under the experimental conditions. Rather, it was only utilized, at low rates, in downstream metabolic processes, e.g., via acetyl-CoA in the citrate cycle and for fatty acid biosynthesis, especially in the F. tularensis subsp. holarctica strains. In summary, the data reflect differential metabolite fluxes in F. tularensis subsp. holarctica and F. novicida suggesting that the different utilization of substrates could be related to host specificity and virulence of Francisella.

Functional Analysis of the Alternative Sigma-28 Factor FliA and Its Anti-Sigma Factor FlgM of the Nonflagellated Legionella Species L. oakridgensis.

Legionella oakridgensis causes Legionnaires' disease but is known to be less virulent than Legionella pneumophilaL. oakridgensis is one of the Legionella species that is nonflagellated. The genes of the flagellar regulon are absent, except those encoding the alternative sigma-28 factor (FliA) and its anti-sigma-28 factor (FlgM). Similar to L. oakridgensis, Legionella adelaidensis and Legionella londiniensis, located in the same phylogenetic clade, have no flagellar regulon, although both are positive for fliA and flgM Here, we investigated the role and function of both genes to better understand the role of FliA, the positive regulator of flagellin expression, in nonflagellated strains. We demonstrated that the FliA gene of L. oakridgensis encodes a functional sigma-28 factor that enables the transcription start from the sigma-28-dependent promoter site. The investigations have shown that FliA is necessary for full fitness of L. oakridgensis Interestingly, expression of FliA-dependent genes depends on the growth phase and temperature, as already shown for L. pneumophila strains that are flagellated. In addition, we demonstrated that FlgM is a negative regulator of FliA-dependent gene expression. FlgM seems to be degraded in a growth-phase- and temperature-dependent manner, instead of being exported into the medium as reported for most bacteria. The degradation of FlgM leads to an increase of FliA activity.IMPORTANCE A less virulent Legionella species, L. oakridgensis, causes Legionnaires' disease and is known to not have flagella, even though L. oakridgensis has the regulator of flagellin expression (FliA). This protein has been shown to be involved in the expression of virulence factors. Thus, the strain was chosen for use in this investigation to search for FliA target genes and to identify putative virulence factors of L. oakridgensis One of the five major target genes of FliA identified here encodes the anti-FliA sigma factor FlgM. Interestingly, in contrast to most homologs in other bacteria, FlgM in L. oakridgensis seems not to be transported from the cell so that FliA gets activated. In L. oakridgensis, FlgM seems to be degraded by protease activities.