AAA+ proteases and their role in distinct stages along the Vibrio cholerae lifecycle

Accepted Manuscript Title: AAA+ proteases and their role in distinct stages along the Vibrio cholerae lifecycle Author: Katharina Pressler Dina Vorkapic Sabine Lichtenegger Gerald Malli Benjamin P. Barilich Fatih Cakar Franz G. Zingl Joachim Reidl Stefan Schild PII: DOI: Reference:

S1438-4221(16)30092-3 http://dx.doi.org/doi:10.1016/j.ijmm.2016.05.013 IJMM 51059

To appear in: Received date: Revised date: Accepted date:

26-2-2016 9-5-2016 24-5-2016

Please cite this article as: Pressler, Katharina, Vorkapic, Dina, Lichtenegger, Sabine, Malli, Gerald, Barilich, Benjamin P., Cakar, Fatih, Zingl, Franz G., Reidl, Joachim, Schild, Stefan, AAA+ proteases and their role in distinct stages along the Vibrio cholerae lifecycle.International Journal of Medical Microbiology http://dx.doi.org/10.1016/j.ijmm.2016.05.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

TITLE AAA+ proteases and their role in distinct stages along the Vibrio cholerae lifecycle.

AUTHORS AND AFFILIATIONS Katharina Pressler, Dina Vorkapic, Sabine Lichtenegger, Gerald Malli, Benjamin P. Barilich, Fatih Cakar, Franz G. Zingl, Joachim Reidl and Stefan Schild*

Institute of Molecular Biosciences, University of Graz, Humboldtstraße 50, A-8010 Graz, Austria

*

Corresponding author. Mailing address: Institute of Molecular Biosciences, University of

Graz, Humboldtstraße 50, A-8010 Graz, Austria. Phone: ++43 (0)316 380-1970. E-mail: [email protected]

ABSTRACT The facultative human pathogen Vibrio cholerae has to adapt to different environmental conditions along its lifecycle by means of transcriptional, translational and post-translational regulation. This study provides a first comprehensive analysis regarding the contribution of the cytoplasmic AAA+ proteases Lon, ClpP and HslV to distinct features of V. cholerae behaviour, including biofilm formation, motility, cholera toxin expression and colonization fitness in the mouse model. While absence of HslV did not yield to any altered phenotype compared to wildtype, absence of Lon or ClpP resulted in significantly reduced colonization in vivo. In addition, a ∆lon deletion mutant showed altered biofilm formation and increased motility, which could be correlated with higher expression of V. cholerae flagella gene class IV. Concordantly, we could show by immunoblot analysis, that Lon is the main protease responsible for proteolytic control of FliA, which is required for class IV flagella gene transcription, but also downregulates virulence gene expression. FliA becomes highly sensitive to proteolytic degradation in absence of its anti-sigma factor FlgM, a scenario reported to occur during mucosal penetration due to FlgM secretion through the broken flagellum. Our results confirm that the high stability of FliA in the absence of Lon results in less cholera toxin and toxin corgulated pilus production under virulence gene inducing conditions and in the presence of a damaged flagellum. Thus, the data presented herein provide a molecular explanation on how V. cholerae can achieve full expression of virulence genes during early stages of colonization, despite FliA getting liberated from the anti-sigma factor FlgM.

KEYWORDS Motility; biofilm; virulence; flagella; mucosal penetration; cholera

INTRODUCTION The facultative human pathogen Vibrio cholerae causes the severe life-threatening secretory diarrheal disease cholera, characterized through a massive loss of water causing rapid dehydration (Koch, 1884). Along its lifecycle, V. cholerae is capable of transiting between two different habitats, the aquatic environment, which acts as a natural reservoir between the epidemic outbreaks, and the gastrointestinal tract of the human host (Nelson et al., 2009; Sack et al., 2004). A key factor for survival and persistence of V. cholerae in the nutrient poor aquatic environment is the ability to form biofilms on chitinous surfaces (Huq et al., 2008; Tamplin et al., 1990). Formation of these bacterial communities is a stepwise process comprising surface adhesion, formation of a monolayer, maturation to a three-dimensional biofilm and finally detachment and release into the planktonic stage (Heithoff and Mahan, 2004; Silva and Benitez, 2016). Not surprisingly, V. cholerae induces a distinct set of genes along the different stages of the biofilm (Moorthy and Watnick, 2005; Schoolnik et al., 2001; Seper et al., 2014). Noteworthy, chitin and nucleic acids are not only the attachment surface or matrix component, respectively, but can also serve as nutrient sources via a set of degradative enzymes and uptake systems, which facilitate the survival fitness of V. cholerae in such nutrient poor conditions (Gumpenberger et al., 2016; Meibom et al., 2004; Pruzzo et al., 2008; Seper et al., 2011). V. cholerae aggregates detached from biofilms might comprise the agent for initial infection via oral ingestion. The cells associated to biofilm clumps might be better protected against acidic conditions, temperature change and high osmolarity, therefore increasing transmission efficiency of the disease (Huq et al., 2008; Tamayo et al., 2010; Zhu and Mekalanos, 2003). Upon entry in the human host, V. cholerae substantially changes its expression profile and induces a set of virulence genes controlled by a complex regulatory network. These changes include the decrease of the second messenger cyclic di-GMP (c-di-GMP), the

activation of ToxR- and TcpP-regulons, which induce transcription of ToxT, the essential transcriptional activator of many virulence genes including the cholera toxin (CTX) and the toxin coregulated pilus (TCP) (Childers and Klose, 2007; Nelson et al., 2009). In addition, V. cholerae uses flagella-dependent motility and mucinases to penetrate through the mucus gel in order to efficiently attach to the epithelial cells in the small intestine (Butler and Camilli, 2005; Freter and Jones, 1976; Freter et al., 1981). The impact of motility is highlighted by reports demonstrating that non-motile V. cholerae mutants colonize 10 to 25 times less efficiently than wildtype (WT) strains (Lee et al., 2001; Liu et al., 2008; Morris et al., 2008). Interestingly studies proved, that the V. cholerae flagella breaks during mucin penetration, which allows secretion of the anti-sigma factor FlgM through its flagellar apparatus (Correa et al., 2004; Liu et al., 2008). This decrease of intracellular FlgM releases the alternative sigma factor FliA, which subsequently will activate FliA-dependent genes (Correa et al., 2004; Liu et al., 2008). Noteworthy, work by Syed et al. demonstrated that FliA can be a potent inhibitor of virulence gene expression. To avoid repression of virulence genes FliA must be effectively inactivated during early stages of colonization to allow full virulence gene expression (Syed et al., 2009). At the late stage of infection V. cholerae alters its gene expression again in order to detach from the epithelium by activating a RpoS-dependent mucosal escape response and to prepare for its transition into the aquatic environment by activating c-di-GMP synthesis and nutrient accumulation (Gumpenberger et al., 2016; Moisi et al., 2013; Nielsen et al., 2006; Schild et al., 2007). In the past, gene expression analyses revealed several interesting and physiological relevant mechanisms for adaptation and fitness of V. cholerae along its lifecycle using a diverse set of reporter-based technologies, microarrays or RNAseq (Bina et al., 2003; Camilli and Mekalanos, 1995; Hsiao et al., 2006; Lee et al., 1999; Mandlik et al., 2011; Moorthy and Watnick, 2005; Nielsen et al., 2006; Papenfort et al., 2015; Schild et al., 2007; Seper et al., 2014). Besides such global changes in the transcriptome, V. cholerae also has to perform

rapid adaptations via posttranslational regulation, e.g. proteolysis.

For example, the

membrane bound transcriptional virulence gene regulators ToxR and TcpP are subjects to regulated intramembrane proteolysis by RseP (YaeL) (Almagro-Moreno et al., 2015a; Almagro-Moreno et al., 2015b; Matson and DiRita, 2005). Furthermore, V. cholerae harbors, as many other bacteria, members of the AAA+ (ATPase associated with a variety of cellular activities) protein family representing proteases active in the cytosol (Neuwald et al., 1999). These include homologs of ClpA/P, ClpX/P, HslU/V (also known as ClpYQ), Lon and FtsH (also known as HflB), which have been best studied in Escherichia coli and generally target misfolded, truncated or mutated proteins and affect the global protein turnover in the cell (Gottesman, 2003; Schmidt et al., 2009). Notably, there is also growing evidence that these proteases have conserved roles in specific, controlled proteolysis in response to environmental stimuli and thereby may contribute to bacterial pathogenesis (Ingmer and Brondsted, 2009). ClpA/P, ClpX/P and HslU/V proteases consist of functional units, with ClpP and HslV being the proteolytic domain, respectively. ClpP, which assembles with two heptameric rings involving the active site, can either associate with the ATPase ClpA or ClpX (Gottesman et al., 1993; Kessel et al., 1995; Maurizi et al., 1998; Wang et al., 1997; Wojtkowiak et al., 1993).

Similarly, the protease HslU/V consists of an ATPase domain (HslU) and a

proteolytic subunit (HslV) (Kessel et al., 1996; Missiakas et al., 1996; Rohrwild et al., 1996). While HslU/V has not been correlated with virulence of bacterial pathogens so far, Clpdependent proteolysis plays several important roles in pathogenesis, especially in Grampositive bacteria via controlling virulence factor production (Ingmer and Brondsted, 2009). For example, in Listeria monocytogenes ClpP degrades an inhibitor of the virulence factor listeriolysin O, which causes food poisoning. In addition a deletion in clpP leads to a decreased ability to multiply in macrophages (Gaillot et al., 2000). In the human pathogen Staphylococcus aureus, which causes a variety of serious infections, a deletion in either clpP

or clpX leads to a reduced transcription of hemolysin α-toxin (Frees et al., 2003; Lowy, 1998). In case of Streptococcus pneumoniae, causing pneumonia, bacteremia and meningitis a clpP mutant is not only deficient in colonization of the nasopharynx and survival in the lungs of mice, but also exhibits shorter survival than the WT in presence of murine macrophages (Kwon et al., 2004). Contrary to the above mentioned AAA+ proteases, FtsH and Lon harbor the ATPase and proteolytic activity on a single polypeptide chain (Langklotz et al., 2012; Licht and Lee, 2008; Park et al., 2006). Furthermore, FtsH is unique as it possesses an inner membrane anchor and has been shown to be essential for viability in E. coli (Langklotz et al., 2012; Ogura et al., 1999). In S. aureus, ftsH mutants exhibit reduced colonization fitness in a murine model and FtsH degrades MgtC in Salmonella, which is required for intramacrophage survival (Alix and Blanc-Potard, 2007; Lithgow et al., 2004). Finally, Lon plays a major role in the protein quality control of the cell by degradation of misfolded or unstable proteins and has also been shown to be important for virulence in several pathogens, including Gram-positive as well as Gram-negative bacteria (Phillips et al., 1984) (Boddicker and Jones, 2004; Cohn et al., 2007; Gottesman, 1996; Takaya et al., 2003). For example, Lon affects the type III secretion system (T3SS), which is used to translocate virulence proteins into host cells.

In Salmonella enterica serovar Typhimurium and

Pseudomonas syringae, Lon acts as negative regulator of the T3SS (Takaya et al., 2005). In contrast, absence of Lon in Yersinia pestis results in repression of T3SS genes, since Lon rapidly degrades YmoA, being a transcriptional repressor of T3SS (Jackson et al., 2004). In Pseudomonas aeruginosa, Lon controls production of homoserine lactones, which mediate quorum sensing, and thereby interferes with virulence gene expression (Takaya et al., 2008). Evidence for the importance of the AAA+ proteases along the V. cholerae lifecycle is scarce. This study investigates the role of the AAA+ proteases of a V. cholerae El Tor isolate in distinct stages of its lifecycle including biofilm formation, motility and colonization fitness.

Our results demonstrate that Lon and ClpP contribute to colonization fitness in vivo. In addition, biofilm formation, motility and CTX expression are significantly altered in a lonmutant. In a more detailed analysis the alternative sigma-factor FliA was revealed to be a target of Lon mediated proteolysis. Finally, the high stability of FliA in the absence of Lon was correlated with altered expression levels of class IV flagellar genes and CTX, providing an explanation of the increased motility and colonization defect of a lon-mutant.

MATERIALS AND MEHODS Bacterial strains and growth conditions. Bacterial strains and plasmids used in this study are listed in Table S1; oligonucleotides are listed in Table S2. V. cholerae SP27459, a spontaneous streptomycin (Sm)-resistant mutant of the clinical isolate O1 El Tor Inaba P27459 was used as WT strain (Pearson et al., 1993). E. coli strains DH5αλpir and SM10λpir were used for genetic manipulations (Hanahan, 1983; Kolter et al., 1978; Miller and Mekalanos, 1988). If not noted otherwise, strains were cultured in Luria Bertani (LB) broth (1% tryptone; 1% NaCl; 0.5% yeast extract), on LB broth agar plates with aeration at 37°C, or for biofilm formation under static conditions at room temperature (RT).

If required,

antibiotics or other supplements were used in the following final concentrations: streptomycin (Sm), 100 μg/ml; ampicillin (Ap), 100 μg/ml or in combination with other antibiotics, 50 μg/ml; chloramphenicol (Cm), 5 μg/ml; isopropyl-β-thiogalactopyranoside (IPTG), 0.5 mM; 5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside (X-Gal), 100 μg/ml; arabinose (Ara), 0.02%; glucose (Gluc), 0.2%; sucrose (Suc), 10%. Construction of in-frame deletion mutants, expression plasmids and reporter fusions. The isolation of chromosomal DNA, PCR reactions, the purification of plasmids or PCR products, the construction of suicide and expression plasmids as well as the subsequent generation of deletion mutants were carried out as described previously (Seper et al., 2011). Qiaquick® Gel extraction and Qiaquick® PCR Purification kits (Qiagen) were used for purifying PCR products and digesting plasmid DNA. PCR reactions for subcloning were carried out using the Q5® High-Fidelity DNA Polymerase (NEB), while Taq DNA Polymerase (NEB) was used for all other PCRs. Deletion mutants were generated using derivatives of the suicide vector pCVD442 in combination with the method described by Donnenberg and Kaper (Donnenberg and Kaper, 1991). Respective suicide vectors were constructed by PCR-amplification of approximately 800 bp fragments, representing upstream and downstream regions of the gene of interest,

using the oligonucleotide pairs x_y_1 and x_y_2 or x_y_3 and x_y_4, where x represents the gene and y the restriction site/enzyme used (Table S2). Subsequently, generated fragments were digested with the appropriate restriction enzyme indicated by the name of the oligonucleotide, and finally ligated into an identically digested suicide plasmid pCVD442. The respective suicide plasmids were first transformed into E. coli Sm10λpir and then transferred into V. cholerae via conjugation. Cells were grown on Sm- and Ap- containing agar plates to select for the integration of the plasmid into the chromosome. This selection was followed by growth on sucrose to obtain Aps colonies, in which an excision of the plasmid from the chromosome took place. The correct deletions were confirmed by PCR (data not shown). GFP expressing strains were generated by insertion of gfp in the lacZ locus using the suized plasmid pJZ111 (Seper et al., 2014). A derivative of pGPphoA was constructed to obtain chromosomal transcriptional fusion of phoA to tcpA, as the phoA acts as useful genetic marker in V. cholerae. A tcpA gene fragment containing the translational stop codon was amplified by PCR using oligonucleotide pair pGPphoA_tcpA_y_fw and pGPphoA_tcpA_y_rev (Table S2), where y represents the restriction site/enzyme. To construct the expression plasmids the respective gene was PCR amplified using the oligonucleotide pairs x_y_fw and x_y_rev, where x represents the gene and y the restriction site/enzyme used (Table S2). In all cases, the respective PCR fragments were digested with the appropriate restriction enzymes and ligated into a identically digested pGPphoA, arabinose-inducible pBAD33, IPTG-inducible pMMB67EH, or IPTG-inducible pFLAG-macTM, to add a N-terminal FLAG-tag. Ligation products were transformed into DH5αλpir and Apr or Cmr colonies were characterized by PCR for the correct constructs (data not shown). For pGPphoAtcpA, chromosomal transcriptional phoA fusions were constructed as described previously using derivates of pGP704 (Seper et al., 2011). Otherwise, plasmids were isolated and transformed into V. cholerae strains, which were then tested for

complementation by swarming assay, performing an immunoblot analysis or static biofilm analysis. Growth kinetics. Growth kinetics were essentially performed as previously described in transparent 24-well plates (Greiner) with 1 ml culture volume (Gumpenberger et al., 2016; Moisi et al., 2013; Seper et al., 2011). Briefly, the respective strains were grown in a preculture over night (ON) in LB with aeration and shaking at 37°C. Cells derived from the precultures were adjusted to OD600 of 0.01 in LB or media reflecting the motility agar composition (1% tryptone; 0.5% NaCl; 0.5% yeast extract) supplemented with arabinose or IPTG to grow them under inducing conditions, respectively. The OD600 was monitored every 30 min in the SPECTROstarNano microplate reader (BMG Labtech) at 37°C or RT with shaking. For presentation of data, at least four independent growth curves were analyzed for each strain tested. The median values were calculated and plotted. Generation of whole cell extracts. For whole cell extracts, appropriate amounts of V. cholerae cultures grown ON were inoculated in fresh LB to an OD600 of 0.1, grown in LB broth to an OD600 of 0.5 following induction with IPTG (0.5 mM) for 4 h. Cell equivalents reflecting 1 ml of an OD600 1.3 culture, were harvested by centrifugation in an Eppendorf centrifuge (5 min at 5,000 g), resuspended in 4x Laemmli buffer, boiled for 30 min at 100°C and either stored at -20°C or directly used for SDS-PAGE. SDS-PAGE and immunoblot analysis. To separate proteins the standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) procedure, in combination with 15% gels and the Prestained Protein Marker Broad Range (New England Biolabs) as a molecular mass standard, was used. Proteins were stained according to Kang et al. (Kang et al., 2002) or transferred to a nitrocellulose membrane (Amersham) for immunoblot analysis, which was performed as described previously (Roier et al., 2012) using mouse anti-Flag antisera (1:5.000 diluted in 10% skim milk, Sigma-Aldrich, F3165-1MG) as primary and peroxidase-conjugated goat anti-mouse (diluted 1:10.000 in 10% skim milk, Dianova GmbH

115-035-003 IgG, Hamburg) as secondary antibody. Loading of equal amounts proteins was always verified by Kang staining following SDS-PAGE in parallel to the immunoblot analysis. Chemiluminescence detection was performed by incubating each membrane in an ECL solution (Bio-Rad Laboratories) with subsequent exposure in a ChemiDoc XRS system (Bio-Rad Laboratories) in combination with the Quantity One software (Bio-Rad Laboratories). Motility assay. The motility assay was performed to compare the swarming ability of WT and mutants. Therefore swarm agar plates (1% tryptone; 0.5% NaCl; 0.5% yeast extract; and 0.3% agar) were used as previously described (Moisi et al., 2009). Plates were inoculated with 2 µl from respective ON cultures, grown in LB broth at 37°C, 180 rpm with aeration and incubated at RT for 18 h, after which time the diameter of growth of the respective strain was measured. Static biofilm assay. Static biofilm assays were performed in microtiter plates and assayed by crystal violet staining as previously published (Seper et al., 2011) with some modifications. Briefly, the respective strains were grown ON on LB-Sm or LB-Ap/Gluc agar plates (for plasmid containing strains), suspended in LB-Sm or LB-Ap/IPTG (for plasmid containing strains), adjusted to an OD600 of 0.013 and inoculated in a 96 well microtiter plate (U bottom, Sterilin) for 48 h. Wells were subsequently rinsed using a microplate washer (Anthos Mikrosysteme GmbH, Fluido2), the biofilm was stained with 0.1% crystal violet, solubilized in 96% ethanol and the OD595 was measured (BGM Labtech SPECTROstarNano) to quantify the amount of biofilm formation. Flow cell biofilm formation, confocal laser scanning microscopy and COMSTAT analysis.

For visualization and quantification of dynamically formed biofilm, the three

channel flow cell system using 2% LB-Sm broth (24 h, RT) was used as described previously (Seper et al., 2011; Seper et al., 2014). The respective GFP expressing V. cholerae strains were used for biofilm formation to allow acquisition of fluorescent images with confocal laser

scanning microscopy.

Images of biofilms were acquired using a Leica SP5 confocal

microscope (Leica Microsystems, Mannheim, Germany) with spectral detection and a Leica HCX PL APO CS 63x water objective (NA 1.2). Optical sectioning was performed in 0.13 µm steps. GFP was excited at 488 nm, fluorescence emission was detected between 500–560 nm, and images were recorded without differential interference contrast (DIC) optics. For visualization and processing of image data the Leica LAS AF Lite and ImageJ 1.46 software was used. Quantification and morphological analysis of image stacks was performed using the computer program COMSTAT (http://www.comstat.dk) (Heydorn et al., 2000; Vorregaard et al.). Alkaline phosphatase (PhoA) and -galactosidase (LacZ) assays. To determine the enzymatic activities for chromosomal vpsA-phoA or plasmid-encoded flrAp-, flrBp-, flaAp-, and flaBp-lacZ transcriptional fusions, alkaline phosphatase or -galactosidase assays were performed as described previously from respective ON cultures (Moisi et al., 2009; Schild et al., 2005b). The activities are expressed in Miller units, given by (A405 x 1000)/(A600 x ml x min). Competition assays. Competition assays for intestinal colonization in infant mice (in vivo) and for growth in LB broth (in vitro) were performed with a mixture of mutant (LacZ+) and isogenic WT strains (LacZ-) to allow differentiation between the strains on X-Gal plates as previously described (Camilli and Mekalanos, 1995; Moisi et al., 2009; Schild et al., 2007). CD-1 mice (Charles River Laboratories) were used in all experiments in accordance with the rules of the ethics committee at the University of Graz and the corresponding animal protocol, which has been approved by the Austrian Federal Ministry of Science and Research Ref. II/10b. Mice were housed with food and water ad libitum and monitored under the care of full-time staff.

6-day old mice were separated from their dams 1 h before infection.

Subsequently, they were anesthetized by inhalation of isoflurane gas and then inoculated by oral gavage with 50 µl of a 1:1 mixture of mutant and isogenic WT strain (approx. 1 x 105

CFU/ mouse). To determine the exact inputs appropriate dilutions of the inocula were plated on LB-Sm/X-Gal plates. After 24 h, the mice were sacrificed and the small intestine from each mouse was collected by dissection. The small intestine was mechanically homogenized in LB broth with 15% glycerol and appropriate dilutions were plated on LB-Sm/X-Gal. In vitro competitions were done by inoculation of 2 ml of LB with ~105 CFU of the inoculum. The cultures were incubated 24 h at 37°C with aeration, subsequently diluted in LB and appropriate dilutions were plated on LB-Sm/X-Gal.

After incubation at 37°C ON, the

colonization rates of mutant and WT were determined by counting the CFU and backcalculation to the original volume of the homogenized small intestine or ON culture. The competitive index (CI) represents the ratio of mutant to WT CFU recovered at 24 h, normalized for the input ratio. CTX ELISA.

CTX production in culture supernatants was determined by the

ganglioside GM1 ELISA (Svennerholm and Holmgren, 1978). V. cholerae strains were grown under virulence gene inducing conditions at 37°C for 4 h anaerobically using AKI broth, followed by 4 h aerobic growth with shaking at 180 rpm (Iwanaga and Yamamoto, 1985; Iwanaga et al., 1986). Shearing of the flagellum was achieved by homogenization of cell culture every 45 min for 45 sec during the aerobic growth using Tissue-Tearor Blade-type Homogenizer (BioSpec) at 32,000 rpm. CFU plating of samples taken before and after treatment with the homogenizer excluded cell lysis or other adverse effects on cell viability. Loss of motility was controlled by microscopy in samples taken right after treatment with the homogenizer. Subsequently, cells were removed from CTX containing supernatants by centrifugation and supernatants were stored at −20°C. ELISA plates (BRANDplates®, 96well, immunoGrade™) were coated with 10 µg/ml GM1 ganglioside (Calbiochem) in 60 mM Na2CO3 ON at 37°C and washed four times with PBS-T pH 7.4. Free binding sites were blocked with 4 mg/ml BSA in PBS (BSA-PBS) for 1 h at RT. After washing as described above, CTX containing supernatants were diluted in PBS and added to the plate.

Additionally, purified CTX in PBS (Sigma-Aldrich) was inoculated in separate wells to generate a standard curve. ELISA plates were incubated with supernatants and purified CTX for 1 h at RT and were washed again as described above. After incubation with the primary antibody (anti-CTX antibody produced in rabbit, Sigma-Aldrich, C3062-1ML), diluted 1∶10,.000 in BSA-PBS for 1 h at RT, ELISA plates were washed four times with PBS-T. After incubation with the secondary antibody (peroxidase conjugated goat anti-rabbit, Dianova GmbH 111-035-003 IgG, Hamburg), diluted 1∶2.000 in BSA-PBS for 1 h at RT and subsequent washing, the ELISA plates were incubated with TMB Substrate Reagent Set (BioLegend, Vienna) for detection of CTX. The reaction was stopped by the addition of 1 M H3PO4 and ELISA plates were measured at OD450 by using SPECTROstarNano microplate reader (BMG Labtech). Live cell microscopy. For live cell microscopy 10 µl of a 1:10 dilution of V. cholerae WT grown under virulence gene inducing conditions were seeded on a micro slide (BRAND® microscope slide 76*26*1 mm; coverglass: 0.13–0.16 mm, VWR) directly before or after treatment with a homogenizer. Movies were recorded with Nikon Eclipse Ti-E confocal microscope, using a Nikon DS-Qi2 camera, Nikon Plan Fluor 40× objective (NA 1.30) and analyzed by Nis-Elements BR version 4.30.02 software. Statistical analysis.

Data were analyzed using the Mann-Whitney U test or a

Kruskal-Wallis test followed by post hoc Dunn’s multiple comparisons. Differences were considered significant at P values of < 0.05. For all statistical analyses, GraphPad Prism version 6 was used.

RESULTS AND DISCUSSION Increased biofilm formation of ∆lon correlates with increased vps expression. To investigate the impact of cytosolic AAA+ proteases on important stages in the V. cholerae lifecycle, non-polar in-frame deletion mutants of lon, clpP and hslV were generated. None of the generated AAA+ protease mutants showed a significant growth defect on LB broth compared to the WT, indicating that these proteases are dispensable for general fitness in nutrient rich media (Fig. S1). Several attempts to obtain a deletion mutant of ftsH failed, suggesting that the inner membrane associated protease FtsH is either essential or fitness of the ftsH-mutants is strongly reduced, which would be consistent with previous reports in E. coli (Langklotz et al., 2012; Ogura et al., 1999). Hence, an ftsH-mutant was not included for phenotypical characterization throughout this study. We characterized the biofilm formation capacity of the mutant strains after 48 h using a static biofilm assay (Fig. 1a). In comparison to the WT, ∆clpP and ∆hslV showed no altered biofilm production, while ∆lon exhibited a significant increase. Biofilm levels even lower to the WT were achieved by expression of lon in trans from an inducible plasmid in the ∆lon mutant, but not by the empty vector control (Fig. 1b). As shown in Figure S2 b, ∆lon plon exhibits comparable growth to WT with empty vector. Thus, a simple growth defect causing such low biofilm levels can be excluded, but induction via IPTG might have caused an overexpression of Lon reducing the biofilm formation even below WT levels. V. cholerae mutants with increased biofilm formation compared to WT have been reported previously. These phenotypes are frequently correlated with enhanced V. cholerae exopolysaccharide (VPS) production, which represents the main component of the extracellular biofilm matrix (Yildiz and Schoolnik, 1999). For example, hapR mutants or genetically engineered strains with increased c-di-GMP levels show high expression levels of the vps genes and consequently increased biofilm production (Tischler and Camilli, 2004; Yildiz et al., 2001; Zhu and Mekalanos, 2003). To elucidate whether the increased biofilm formation of the lon

mutant can be correlated with enhanced vps expression, a chromosomal transcriptional vpsAphoA fusion was introduced into WT and ∆lon. The vpsA gene is the first gene in the vps-I locus, hence, the measured PhoA-activity reflects the transcriptional levels of vpsA (Fong et al., 2010; Seper et al., 2014). Since ∆lon showed a significant increase in PhoA-activity compared to the WT (Fig. 1c), the enhanced biofilm formation of ∆lon under static conditions can be at least partially attributed to higher expression levels of vps genes. In contrast to our results obtained under static conditions, a recent study reported decreased biofilm of a V. cholerae lon mutant in a dynamic setup, which could be linked to lower c-di-GMP levels and downregulation of vps genes (Rogers et al., 2016).

Intrigued by this discrepancy, we

additionally performed flow cell biofilms to investigate the dynamic biofilm formation of the WT and ∆lon used in our study. Consistent with our observations under static conditions, ∆lon exhibited higher biomass and thickness compared to the WT under dynamic conditions (Fig. 2, Fig. S3). So far and considering the Rogers et al. study, we can only speculate, why such inverse phenotypes regarding biofilm formation and vps expression of lon-mutants in V. cholerae strains exist. As both studies used different V. cholerae strains, one could speculate that Lon might have distinct activities in V. cholerae isolates. Such isolate-specific Lon phenotypes need to be comprehensively investigated in future studies including more V. cholerae isolates and comparative analyses of biofilm formation. Deletion of lon affects motility in V. cholerae. Next, motility was investigated on swarm agar plates.

As all strains tested for swarming exhibited similar growth under

conditions reflecting the motility assays (Fig. S2), the swarming phenotypes observed are not simply due to growth defects. Comparing the swarming behavior of the protease mutants to the WT, ∆lon shows a pronounced hypermotile phenotype (Fig. 3a). By measuring the diameter of the migration zone we could identify an almost two-fold significant increase in the swarming diameter of ∆lon in comparison to the WT, whereas the other proteases showed

no difference (Fig. 3b). The hypermotile phenotype of ∆lon could be restored to WT levels by expression of lon in trans (Fig. 3c, d). Similar phenoytpes of lon mutants have been observed in other bacteria, e.g. Proteus mirabilis and Vibrio parahaemolyticus, and recently have also been reported for another V. cholerae isolate indicating that this might be a globally conserved regulatory mechanism (Clemmer and Rather, 2008; Rather, 2005; Rogers et al., 2016; Stewart et al., 1997). In more detail, Lon of P. mirabilis was found to target FlhD, an activator of the flagellar gene cascade (Clemmer and Rather, 2008).

Similarly, Barembruch and Hengge demonstrated that

expression levels of the alternative sigma factor FliA are controlled by FlgM-modulated Lon proteolysis in E. coli K12 (Barembruch and Hengge, 2007). V. cholerae also encodes FliAand FlgM-homologs. Notably, FlgM of V. cholerae has already been shown to physically interact with FliA (Correa et al., 2004). As an alternative sigma factor, FliA of V. cholerae is required for transcription of class IV flagellar genes encoding flagellins and the motor components (Prouty et al., 2001). In order to investigate, whether this regulatory circuit comprising FliA, FlgM and Lon might also be active in V. cholerae, we constructed non-polar the deletion mutants ∆fliA, ∆flgM and a ∆fliA∆flgM double mutant as well as appropriate, combinable expression plasmids and analyzed the swarming behavior on motility plates (Fig. 4a - f). Consistent with previous reports from the classical biotype (Correa et al., 2004), deletion of fliA or flgM in the herein used V. cholerae El Tor isolate resulted in strains with significantly reduced motility, which could be at least partially complemented by expression of the respective gene in trans (Fig. 4a-d). Furthermore, the non-motile phenotype of a ∆fliA∆flgM could be partially restored by combined expression of fliA and flgM in trans, but not by expression of fliA alone (Fig. 4e, f). Utilizing the ∆fliA pFLAGfliA and ∆fliA∆flgM pFLAGfliA strains expressing FLAGtagged FliA, which allows detection via immunoblot analysis and subsequent densitrometric evaluation of FLAG-FliA intensities, we investigated the stability of FLAG-FliA in presence

and absence of FlgM (Fig. 5a). A robust band corresponding to the size of FLAG-FliA was detected in whole cell extracts of ∆fliA pFLAGfliA, but neither in whole cell extracts of the negative control ∆fliA pFLAG nor of ∆fliA∆flgM pFLAGfliA. This suggests that the absence of FlgM destabilizes FLAG-FliA in V. cholerae and results in rapid proteolysis. Using ∆fliA∆flgM pFLAGfliA as a parental strain, we deleted lon, clpP and hslV and screened for re-appearance of a FLAG-FliA band in whole cell extracts, which would indicate a stabilization of FLAG-FliA. While deletion of hslV only resulted in a FLAG-FliA band of low intensity, the deletion of clpP and primarily the deletion of lon restored the FLAG-FliA band to WT levels (5a). Consistent with E. coli, these results suggest that in V. cholerae FlgM can protect FliA from proteolytic degradation, which is mainly mediated by Lon and to a lesser extent by ClpP. In this line, deletion of lon in addition to flgM restored the motility defect of ∆flgM almost to WT levels (Fig. 4g, h), whereas deletion of clpP in ∆flgM did not alter motility (Fig S4a, b). These results reinforce the observation that the driving force of FliA proteolysis is Lon. Having identified Lon and ClpP targeting FliA in absence of FlgM, we asked whether these proteases might also contribute to proteolysis of FLAG-FliA in presence of FlgM. Hence, deletions of lon or clpP were generated in the background of ∆fliA pFLAGfliA and whole extracts analyzed by immunoblot for abundance of Flag-FliA (Fig. 5b). While absence of ClpP in this background did not strongly affect the intensity of the FLAG-FliA band, the absence of Lon resulted in a slightly more intensive band compared to the whole cell extracts of ∆fliA pFLAGfliA, which served as a control. This suggests that Lon mediates proteolytic degradation of FliA at a basal level also in presence of FlgM. Collectively, these data identify Lon as the FliA degrading protease under FlgM depleted conditions. Lon affects transcription of FliA-dependent flagellar class IV genes. The relative high abundance of FliA in the absence of Lon, might trigger an increased expression of FliAdependent genes, which might lead to the observed hypermotile phenotype. In V. cholerae

the flagellar gene transcription is strictly organized and controlled within a transcription hierarchy of four classes, with class II and III being RpoN-dependent and class IV being FliAdependent (Prouty et al., 2001). To evaluate the impact of Lon on the transcription of all four classes, we measured expression levels of one gene from each class, using representative plasmids with flagellar gene promotor-lacZ transcriptional fusions in the WT and the ∆lon background (Fig 6). While no difference for the transcription of the flagellar gene classes I to III between WT and ∆lon was observed, ∆lon showed a significant higher transcription of class IV flagellar genes (Fig 6). In summary, this data correlates with the increased stability of FliA and upregulated expression levels of class IV flagellar genes in absence of Lon and may allow an explanation of the observed hypermotile phenotype of ∆lon. Lon and ClpP contribute to in vivo colonization fitness. To provide a physiological context, the impact of the three AAA+ proteases on in vivo colonization, using the infant mouse model, was investigated.

The competition experiment was conducted using the

respective deletion mutant (lacZ+) and a fully virulent lacZ- derivative of the WT in a 1:1 ratio. It is well established that deletion of lacZ does not impact colonization of the mouse intestine and lacZ- derivatives of the V. cholerae WT used within this study have previously been used in such competition assays (Nesper et al., 2000; Schild et al., 2005a). In the case of ∆lon and ∆clpP the mutants exhibited already a minor defect during an in vitro competition in regular LB broth (Fig. 7a, b). However, in both cases the fitness disadvantage was even more pronounced during the in vivo colonization reflected by a significant attenuation in vivo compared to the in vitro control condition. No attenuation was observed for ∆hslV under both conditions tested (Fig. 7c). These data provide an insight into the importance of two major proteases for in vivo survival. Lon affects virulence gene expression. Based on the observed in vivo defects, altered expression of virulence factors, e.g. CTX, was investigated for ∆lon, ∆clpP and ∆hslV using a well-established growth condition for the induction of virulence genes in V. cholerae

El Tor based on AKI broth (Iwanaga and Yamamoto, 1985; Iwanaga et al., 1986). While ∆clpP and ∆hslV produced similar CTX levels compared to the WT, ∆lon showed a significant increase in CTX production (Fig. 8a), which is consistent with a recent study reporting increased virulence gene expression of a lon-mutant in a different V. cholerae isolate (Rogers et al., 2016). We can only speculate about the cause of the increased CTX expression levels in ∆lon, but it should be noted that deletion of AAA+ proteases can have pleiotropic effects and obviously requires further rigorous and comprehensive investigation of the relevant regulatory pathways involved.

Intriguingly, hypermotility and increased

virulence gene expression of ∆lon should rather be attributed to strains with enhanced colonization fitness (Butler and Camilli, 2004; Butler et al., 2006; Merrell et al., 2002). On the other hand, Syed et al. demonstrated that FliA is a potent inhibitor of V. cholerae virulence gene expression, e.g. CTX and TCP (Syed et al., 2009).

In V. cholerae the

flagellum breaks during mucosal penetration and releases FlgM through the damaged flagellum (Correa et al., 2004; Liu et al., 2008). Concordantly, ∆flgM, a mutant with rapid FliA proteolysis and consequently low cytoplasmic FliA levels (Fig. 5a), demonstrated significantly higher CTX expression compared to WT (Fig. 8a). Based on these findings the liberated FliA of V. cholerae cells penetrating through the mucus should be rapidly eliminated to allow proper virulence gene expression. In order to mimic the breakage of the flagellum during mucosal penetration, we treated cultures growing under virulence gene inducing conditions with a homogenizer, which shears off the flagellum and turns the majority of the bacterial population into non-motile bacteria (Movie S5 and S6). CFU plating of samples taken before and after treatment with the homogenizer excluded cell lysis or other adverse effects on cell viability (Fig. S7). Evaluation of the CTX expression levels under these conditions revealed that WT and ∆flgM produce similar levels of CTX, while ∆lon shows significantly less CTX production (Fig. 8b).

In addition, chromosomal tcpA-phoA

transcriptional fusions in WT, ∆lon and ∆flgM were constructed to determine the expression

levels of the major subunit of the TCP, representing another virulence and major colonization factor. Concordant with the CTX results described above and the study by Rogers et al. (Rogers et al., 2016), ∆lon showed significantly increased TCP expression compared to WT (Fig. 9a).

In further consistenty to our observations, treatment with a homogenizer,

mimicking breakage of the flagella, resulted in significantly deacresed PhoA activites in ∆lon compared to WT (Fig. 9b). Thus, it can be hypothesized that breakage of the flagellum during mucosal penetration allows secretion of FlgM and liberates FliA, which gets rapidly degraded via Lon. In absence of Lon, FliA is rather stable and inhibits full virulence gene expression, which would provide an explanation for the observed in vivo defect of ∆lon. Conclusion. In summary, the characterization of ∆lon, ∆clpP and ∆hslV revealed that AAA+ proteases mainly contribute to phenotypical changes in the various stages along the V. cholerae lifecycle. While we could not detect any altered phenotype for ∆hslV compared to the WT under the conditions tested, absence of ClpP significantly reduced colonization fitness of V. cholerae in vivo. Consistent with this observation clpP-mutants have been reported to negatively affect virulence in other bacteria, e.g. S. aureus, L. monocytogenes and Salmonella ssp. (Frees et al., 2003; Gaillot et al., 2000; Tomoyasu et al., 2002; Tomoyasu et al., 2003). The exact role of ClpP during in vivo colonization needs to be investigated in future studies, but based on our results we can at least conclude that ClpP has impact on CTX expression. The most versatile effects during this study have been observed for ∆lon, which showed increased static and dynamic biofilm formation, increased motility, less colonization fitness and altered CTX and TCP expression. Thus, Lon contributes to several steps along the V. cholerae lifecycle. Based on recent work from E. coli (Barembruch and Hengge, 2007), the alternative sigma-factor FliA was shown to be a proteolytic target of Lon. The data presented herein, correlate the increased motility of ∆lon with enhanced FliA stability and higher expression levels of class IV flagellar genes. In addition, our data indicates that FliA can be protected from rapid proteolysis by the anti-sigma factor FlgM. Thus, liberation of FliA from

FlgM can have two consequences: either FliA gets associated with the RNA-Polymerase and enables expression of FliA-dependent genes or FliA is degraded. Considering recent reports, substantial FliA liberation takes place in an early stage of infection, when V. cholerae needs to penetrate through the mucosal layer (Correa et al., 2004; Liu et al., 2008). The resulting breakage of the flagellum allows FlgM-secretion, while the remaining FliA is a potent inhibitor of virulence genes (Syed et al., 2009). So far a missing link was the effective and prompt removal of FliA to allow full virulence expression, which can now be explained by rapid proteolysis mainly mediated by Lon.

ACKNOWLEDGEMENTS The work was supported by the Austrian Science Fund (FWF) grants: W901 (DK Molecular Enzymology) to K. P., D. V., F. C., F. G. Z. and S. S. as well as P27654 to S. S..

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FIGURE LEGENDS Fig. 1: Δlon shows increased static biofilm formation and increased vpsA expression compared to the WT. (a,b) Biofilm formation of the WT, deletion mutants of the AAA+ proteases (a) as well as WT with control plasmid (p stands for pMMBEH67, see table S1 for details) or complemented mutant (b), as indicated, were quantified after 48 h. The biofilm formation capacity was assayed under static conditions by crystal violet staining and subsequent determination of the OD595. independent measurements.

Shown are the medians from at least eight

The error bars indicate the interquartile range.

Significant

differences (* P < 0.05 Kruskal-Wallis test followed by post hoc Dunn’s multiple comparisons) are indicated.

(c) Alkaline phosphatase activities (in Miller Units) were

measured from ON cultures of the WT and ∆lon with a chromosomal vpsA-phoA transcriptional fusion. Shown are the medians from at least six independent measurements. Significant differences between the data sets are marked by asterisks (P < 0.05 Mann– Whitney U test).

Fig. 2: Absence of Lon results in alteration of the biofilm architecture. Image stacks of WT and ∆lon biofilms were analyzed for biomass (a), the maximum thickness (b), the roughness coefficient (c) and the average thickness (d) using the COMSTAT software (Heydorn et al., 2000). Shown are the medians of at least fifteen image stacks from nine independent experiments for each strain. The error bars indicate the interquartile range. Significant differences (* P < 0.05 Mann–Whitney U test) are indicated.

Fig. 3:

Δlon exhibits increased motility compared to the WT.

(a, c)

Shown are

representative images of swarm plates highlighting the motility phenotypes of the WT, deletion mutants of AAA+ proteases (a) as well as WT with control plasmid (p stands for pMMBEH67, see table S1 for details) or complemented mutant (c). Genetic backgrounds are

indicated above. (b, d) Swarming diameters of the respective strains are indicated. Shown are the medians from at least eight independent measurements. The error bars indicate the interquartile range. Significant differences between the data sets are marked by asterisks (P < 0.05 Kruskal-Wallis test followed by post hoc Dunn’s multiple comparisons).

Fig. 4: Combined presence of FliA and FlgM is essential for motility. (a, c, e, g) Shown are representative images of swarm plates highlighting the motility phenotypes of WT and mutants with respective control plasmids or mutants with expression plasmids. Genetic backgrounds are indicated above. (b, d, f, h) Swarming diameters of the respective strains are indicated. Shown are the medians from at least eight independent measurements. The error bars indicate the interquartile range. Significant differences between the data sets are marked by asterisks (P < 0.05 Kruskal-Wallis test followed by post hoc Dunn’s multiple comparisons).

Fig. 5:

Proteolytic degradation of FliA is mainly contributed by Lon and ClpP.  

Immunoblot analysis of whole cell extracts obtained from ∆fliA or its derivatives harbouring either pFLAG or pFLAGfliA (expressing FLAG-FliA). Genetic backgrounds are indicated above. The arrow indicates the detected FLAG-tagged FliA predicted to be approx. 28 kDa. Semiquantitative densitometric evaluation of detected Flag-FliA was performed with the Quantity One software (Bio-Rad Laboratories) and is indicated below the representative immunoblots as arbitrary intensity units (AIU) of detected FLAG-FliA normalized to the ∆fliA pFLAGfliA control sample, which was always set to 1. At least seven independent whole cell extracts of each strain were analyzed. The data is given as median with maximum (superscript) and minimum (subscript).  

Fig. 6: Expression of flagella genes in the WT and the lon mutant. β-galactosidase activities, shown in Miller units, were measured for the WT (open bars) and Δlon (grey bars) carrying the flagellar gene promoter -lacZ fusion plasmids pKEK73 (flrAp-lacZ), pKEK72 (flrBp-lacZ), pKEK80 (flaAp-lacZ) or pKEK79 (flaBp-lacZ). The respective promoter-lacZ fusion as well as the flagellar gene class is indicated on the x axis. Values are the medians from at least eight independent measurements. The error bars show the interquartile range with asterisks indicating significant different medians of activities of WT and Δlon carrying the same plasmid (P < 0.005, Mann-Whitney U test).

Fig. 7: Colonization fitness of V. cholerae AAA+ protease mutants. Results are shown as competitive index (CI) for competition of ∆lon (a), ∆clpP (b) and ∆hslV (c) to a fully virulent LacZ- derivative of the WT in LB broth (in vitro, grey circles) and in vivo using the infant mouse model (black circles). Each circle represents the CI from a single assay. Horizontal bars indicate the median of each data set.

The asterisks indicate significantly different

medians of the in vivo compared to the respective in vitro data set (P < 0.005, using a MannWhitney U test).     Fig. 8:  CTX production of V. cholerae AAA+ protease mutants and ∆flgM.  Shown is the CTX production of V. cholerae WT and mutants as indicated before (a) and after (b) treatment with homogenizer, representing bacterial populations with intact and broken flagella, respectively. Strains were grown under virulence gene factor expressing conditions to monitor CTX production. measurements.

Values are the medians from at least 16 independent

The error bars indicate the interquartile range with asterisks indicating

significant different medians between WT and mutants (P < 0.005, Kruskal-Wallis test followed by post hoc Dunn’s multiple comparisons).

Fig. 9:  Expression levels of tcpA in V. cholerae WT , ∆lon

and ∆flgM.    Alkaline

phosphatase activities (in Miller Units) were measured from cultures of the WT, ∆lon and ∆flgM with a chromosomal tcpA-phoA transcriptional fusion. Strains were grown under virulence gene factor expressing conditions and treated like CTX producing strains (mentioned above). Shown are the medians from at least six independent measurements. Significant differences between the data sets are marked by asterisks (P < 0.05 Mann– Whitney U test).

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