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Genetic requirements and transcriptomics of Helicobacter pylori biofilm formation on abiotic and biotic surfaces

Nov 27, 2020

npj biofilms and microbiomes Abstract Biofilm growth is a widespread mechanism that protects bacteria against harsh environments, antimicrobials, and immune responses. These types of conditions challenge chronic colonizers such as Helicobacter pylori but it is not fully understood how H. pylori biofilm growth is defined and its impact on H. pylori survival. To provide insights into H. pylori biofilm growth properties, we characterized biofilm formation on abiotic and biotic surfaces, identified genes required for biofilm formation, and defined the biofilm-associated gene expression of the laboratory model H. pylori strain G27. We report that H. pylori G27 forms biofilms with a high biomass and complex flagella-filled 3D structures on both plastic and gastric epithelial cells. Using a screen for biofilm-defective mutants and transcriptomics, we discovered that biofilm cells demonstrated lower transcripts for TCA cycle enzymes but higher ones for flagellar formation, two type four secretion systems, hydrogenase, and acetone metabolism. We confirmed that biofilm formation requires flagella, hydrogenase, and acetone metabolism on both abiotic and biotic surfaces. Altogether, these data suggest that H. pylori is capable of adjusting its phenotype when grown as biofilm, changing its metabolism, and re-shaping flagella, typically locomotion organelles, into adhesive structures. Introduction Helicobacter pylori is a significant human pathogen that is difficult to cure. H. pylori infects more than half of the world’s population, making it one of the most common bacterial infections 1 . H. pylori causes chronic gastritis, duodenal and gastric ulcers, and significantly increases the risk of gastric adenocarcinoma and mucosal-associated-lymphoid type (MALT) lymphoma. H. pylori disease is significant: Gastric cancers kill over 700,000 people per year, and peptic ulcer disease has an estimated cost of six billion dollars in the United States alone 1 , 2 . Many of these diseases could be prevented by curing the underlying H. pylori infection 1 , 3 . Antibiotics are used to cure H. pylori infections, but successful treatment remains challenging. The standard of care is a 2-week course of a combination of a proton pump inhibitor and two-three antibiotics (clarithromycin, metronidazole, amoxicillin, or tetracycline), but this treatment leaves ~25% of people uncured 4 , 5 . It is not yet fully understood why cure is so difficult, because H. pylori strains are not typically multidrug resistant. Rising resistance to single antibiotics, especially clarithromycin and metronidazole, is a growing concern and certainly accounts for some treatment failure 5 , 6 . An additional explanation may be that some H. pylori are conditionally antibiotic tolerant due to their growth state and concomitant gene expression. One growth state associated with antibiotic tolerance is biofilm growth 7 , 8 , 9 , 10 . H. pylori can grow as biofilm in vitro 11 , 12 , 13 , 14 , 15 , 16 and in vivo 17 , 18 , 19 . Recent studies have suggested that biofilm-grown H. pylori are tolerant to the commonly used antibiotics clarithromycin, amoxicillin, and metronidazole, and express high levels of putative antibiotic efflux pumps 20 , 21 , 22 . These findings suggest that biofilm formation could be a contributor to H. pylori persistence and the difficulty in curing this infection. Studies on H. pylori biofilms have been relatively few, but recent contributions from high throughput ‘omics’ strategies such as genomics, transcriptomics, and proteomics have identified several biofilm properties 16 , 23 , 24 , 25 . Indeed, H. pylori biofilm are less metabolically active than planktonic cells, possess low protein translation, and display multiple aspects of stress responses 16 . Biofilms have been shown to rely on membrane proteins, genes products of the cytotoxin-associated gene pathogenicity island (cagPAI), which express a type IV secretion system (cag-T4SS) 23 , 24 , and flagellar genes 16 . Flagellar filaments have been shown to be part of the H. pylori biofilm matrix along with proteins, eDNA, carbohydrates, and LPS 16 , 25 , 26 , 27 . Nevertheless, H. pylori biofilm studies have been slowed in part by the fact that H. pylori strains are highly variable. Several lab strains have become model systems in general, however, due to their properties such as ease of growth, solid genetics, or ability to reliably infect mice. One of these is H. pylori strain G27. H. pylori G27 is known for its relatively straightforward genetics and reliable growth. Several recent papers have used H. pylori G27 to document important aspects of H. pylori biofilm formation. These insights include that G27 forms biofilms on many abiotic and biotic surfaces under standard lab conditions, has a matrix composed mainly of proteins but also some eDNA and carbohydrates, that conditions such as lowered Zn- and Mn- promote biofilm formation in part due to LPS modifications, and that AI-2 quorum sensing and the ArsRS regulatory system are important players 14 , 26 , 28 , 29 , 30 . However, the genetic pathways for biofilm formation have not yet been characterized in any H. pylori strain, including G27. As the next logical step in characterizing H. pylori biofilm formation, we undertook studies to further H. pylori G27 as a model system. Here, we characterize H. pylori G27’s biofilm formation on abiotic and biotic surfaces, and perform genomic and transcriptomic approaches to unravel genes associated with the biofilm mode of growth in this strong biofilm-forming H. pylori. Results H. pylori strain G27 forms large-mass biofilms under standard laboratory conditions Multiple H. pylori strains form biofilms, including the H. pylori model strain G27 12 , 29 , 30 , 31 , 32 . For the most part, these studies have used various media and conditions, and not compared the strains side-by-side. Therefore, we first compared the biofilm-forming ability of strain G27 with several H. pylori reference and clinical strains. We used the microtiter plate biofilm assay, referred to as abiotic, with 3 days of growth in Brucella Broth with 10% heat-inactivated fetal bovine serum (BB10), no antibiotics, and microaerobic conditions (10% CO2, 5% O2, 85% N2). After incubation, adherent cells were washed and stained with crystal violet for quantification. All strains adhered to the plastic surfaces at the bottom of the well, and formed crystal-violet stained biomass (Fig. 1 ). Some strains, including G27, also formed biofilms at the air–liquid interface as rings on the plastic and/or a thin, fragile, floating pellicle, as previously noted 28 . Quantification of the adherent biofilms showed that some strains displayed a large amount of biofilm mass, and others displayed less (Fig. 1 ). Lab strains G27, X47AL, and CPY3401 all formed high amounts of biofilm, while others formed low amounts, including the common mouse-infecting H. pylori strain SS1. Varied biofilm biomass was also displayed by clinical isolates, suggesting this is a strain-specific property and not associated with lab culture (Fig. 1 ). Time course analysis suggested that biofilm mass was detectable at 24 h, and steadily increased to a maximum at 3 days of growth (Supplementary Fig. 1A and ref. 26 ). Biofilm formation was influenced by some environmental parameters including pH: the biomass decreased as the pH was lowered from neutral to pH 3 (Supplementary Fig. 1B ). Overall, these experiments show that H. pylori G27 is a type of H. pylori strain that forms a high level of biofilm biomass under lab conditions, with three days of lab culture resulting in maximum biofilm formation. Fig. 1: Specific H. pylori strains form biofilms in rich media. Biofilm formation of H. pylori strains was assessed using the microtiter plate crystal violet biofilm assay. Strains were grown for three days in BB10, with no shaking, under 10% CO2, 5% O2 and 85% N2. Results represent the crystal violet absorbance at 595 nm, which reflects the biofilm biomass. Experiments were performed three independent times with at least six technical replicates for each. Error bars represent standard error of the mean. Discussion We report here characterization of biofilm formation in H. pylori, using strain G27, which forms robust biofilms under many conditions. We identified genes required for and upregulated during biofilm growth. Several of these genes matched previous reports, but new properties were also shown to be key for biofilm formation, including acetone metabolism, hydrogen utilization, DNA methylation, and multiple genes whose products have unknown functions. Overall, our results suggest several universal aspects of H. pylori biofilm growth important on multiple surfaces, as well as properties that appear to vary in importance across models. H. pylori G27 forms robust biofilms that consist of reproducible 3-dimensional and dense packed-cell structure under standard H. pylori growth conditions. Numerous studies support that H. pylori G27 forms biofilms on abiotic surfaces 14 , 16 , 26 , 29 , 30 , 50 , and here we expand those studies to biofilms on epithelial cells, referred to as biotic surfaces. The biofilms formed on biotic surfaces were similar to those on abiotic surfaces in that there were layers of bacteria that were tightly packed and enmeshed in flagellar filaments (Fig. 3 ). Biotic biofilms differed in that the cells were mostly spiral or rod-shaped (Fig. 3 ), versus nearly fully coccoid in the abiotic biofilms (Fig. 2 ). This appearance is consistent with the idea that the abiotic biofilms are under nutrient depletion stress; previous work showed that planktonic cells cultured for this time were also mostly coccoid 16 . Indeed, previous work characterized growth of an H. pylori G27 derivative on epithelial cells, finding the bacteria grew while directly attached into microcolonies that consisted of tightly packed spiral-shaped cells 33 , 34 . These authors elegantly showed that the H. pylori obtain nutrients directly from the cells, including iron, a finding that agreed well with previous reports 51 . Latter work also showed H. pylori G27 formed cell-associated microcolonies 29 . Initially, these microcolonies were not characterized as biofilms 33 , 34 , but we propose that they are in line with Anderson et al. 28 . This idea is based on the fact that there are multiple layers of tightly packed bacteria, attached to a surface and each other, with flagella filament as extracellular structures promoting adherence. These results, in summary, support that H. pylori G27 is an excellent model for both abiotic and biotic biofilm formation. One finding from this work is confirmation that flagella are key for multiple types of H. pylori biofilms, consistent with previous findings on abiotic surfaces 16 , 38 and also studies with E. coli 52 , 53 . Both biotic- and abiotic-surface biofilms contained a meshed network of flagella filaments that promoted biofilm formation, likely playing a structural role to hold bacteria together and to the surface. Mutants lacking flagella showed poor epithelial cell adherence and biotic biofilm formation. In comparison, mutants that had non-functional flagella formed significantly more biofilm compared to isogenic Fla− cells, but did not match wild-type H. pylori. This outcome suggests that active motility is required for full adherence and biofilm formation in this model. Consistent with the importance of flagella, biofilm cells over-expressed flagellar genes: here we show rpoN and flaA, while previous work reported additional flagellar genes 16 . Furthermore, two of eight biofilm-defective transposon mutants affected flagella, faaA and fliK. FaaA encodes a large 348 kD protein of the autotransporter family predicted to have a beta helix structure that would extend substantially from the cell surface 46 . FaaA localizes to the flagellar sheath is plays a role in flagellar stability 46 . Therefore, the faaA mutant may have biofilm defects due to loss of flagella or to other properties of FaaA. FliK is a hook-length control protein, important for normal flagella formation. Mutants lacking fliK form structures that consist of long flagellar hooks, so-called polyhooks 45 , and our results suggest these hooks are not adequate to promote biofilm formation. Our studies thus support that flagella continue to be expressed in H. pylori biofilms and contribute to their formation on both abiotic and epithelial surfaces. Our biofilm-defective transposon mutant screen found evidence that both acetone and hydrogen utilization are important for biofilm formation. The acxA transposon mutant and acxAB defined mutants showed a significant defect on both abiotic and biotic surfaces (Figs 5 , 7 , and 8 ), and the acxABC operon was significantly upregulated in biofilm cells (Table 2 and Supplementary Table 2 ). These gene products are predicted to catalyze the ATP-dependent carboxylation of acetone to acetoacetate, which creates two molecules of acetyl-CoA 44 . The H. pylori acetone carboxylase shows high-sequence identify (59–68% amino acid identity) to characterized acetone carboxylases, and thus is predicted to share the same function. Several microbes use acetone as a carbon and energy source, although this has not been shown for H. pylori. H. pylori acxABC contributes to gastric colonization, in mice and there is acetone in the gastric tissue 44 . Additional work will be needed, however, to characterize the acetone source during biofilm growth and confirm the function of these gene products. Another alternative type of biofilm-related metabolism we found here in multiple ways is the use of hydrogen as an electron donor via H. pylori’s Ni-Fe hydrogenase. Hydrogenase is created by the products of the hydABCDE, hypA, hypBCD, and hypEF operons. hydAB encodes the main hydrogenase subunits, hydC encodes the cytochrome b subunit, and hydD is required for hydrogenase maturation. hydE has an unknown function in H. pylori, but it is known that hydE mutants lose hydrogenase activity, are not rescued by exogenous nickel, and have normal urease activity 48 . Thus, hydE mutants lack hydrogenase function, and because hydE is the last gene in the operon, mutations should not have polar effects. The five hyp genes (hypA-F) are required for cofactor maturation in support of hydrogenase and/or urease. HypE and HypF are required only for hydrogenase activity 48 , 54 , and in heterologous systems, produce the non-protein CN and CO ligands that hold the hydrogenase Fe 55 . The hypE mutant hydrogenase phenotype is corrected by exogenous nickel, but it is not known if the hypF mutant shares this property 48 . These results thus suggest that hypF mutants may have conditional loss of hydrogenase activity, recovering under high Ni. Our results found that hydE mutants were strongly defective in abiotic biofilms, while hypF mutants were more defective in biotic biofilms. It’s difficult to speculate why these differences exist in part because neither of these encoded proteins are well characterized in H. pylori. The results do, however, suggest that hydrogenase activity plays a role in biofilm formation and that some different properties may be needed between these two types of biofilms. Biofilm H. pylori G27 displayed a distinct transcriptome, similar to that reported for strain SS1 16 . Several types of genes were similarly regulated, such as downregulation of TCA cycle genes and upregulation of flagellar genes. There was, however, minimal overlap in the exact genes that were differentially expressed in the biofilms of the two strains. This discrepancy may be due in part to the experimental design differences, in that the SS1 study employed planktonic and biofilm cells in the same culture wells, while the work here used different culture conditions because of the soft G27 pellicle that could not be easily separated during sample preparation. One gene, lptB, was upregulated ∼3-fold in both SS1 and G27 biofilms. lptB encodes a lipopolysaccharide export system ATPase, agreeing well with the observation that LPS components are part of H. pylori biofilm matrix 11 , 30 . Two additional LPS-related genes were upregulated in these studies: G27 rfaJ-1, which encodes an α-1,6-glucosyltranferase that plays an integral role in the biosynthesis of the core LPS 56 , and SS1 lpxB, which encodes a lipid-A-disaccharide synthase 16 . Previous work similarly highlighted the importance of LPS, finding that loss of some LPS-related genes, lpxF and lpxL, promoted biofilm formation 30 . The expression of the gene encoding for LpxF was downregulated in this study. Overall, these results show that LPS regulation is a key aspect of H. pylori biofilms. H. pylori G27 biofilm cells displayed pronounced metabolic changes, including downregulation of glycolysis, the TCA cycle, and specific electron transport chain components. There were seven downregulated genes spread throughout the central metabolic pathways. These included the glycolytic enzyme glucokinase (glk); the TCA enzymes citrate synthase (gltA), isocitrate dehydrogenase (icd), and fumarase (fumC); the TCA-related enzyme alpha-ketoglutarate oxidoreductase (oorD), which catalyzes the conversion of α-ketoglutarate to succinyl coenzyme A; and the electron transport chain components NADH-ubiquinone oxidoreductase (nqo10) and NADH oxidoreductase I (nuoB) (Table 3 and Supplementary Table 3 ). Genes related to energy metabolism, including ferredoxin (HPG27_256), thioredoxin (trxA), and the super dismutase sodB were among the most downregulated genes in our biofilm cells. Interestingly, these same genes were also downregulated in other studies, after adhesion to gastric cells in vitro 42 . Altogether, these data suggest that cells grown in biofilm may decrease flux through glycolysis and the TCA cycle as a way to lower or alter metabolism. This lowered growth state may also contribute to antibiotic tolerance, as described for other organisms 7 , 8 , 9 , 10 . In addition to the pathways described above, the transcriptomics analysis highlighted several other interesting gene expression patterns of H. pylori G27 biofilm cells. The first was upregulation of the cag type IV secretion system (cag-T4SS) genes. H. pylori G27 biofilms expressed high amounts of the genes for the CagB ATPase, and Cag4, encoding a cell wall hydrolase of the cag T4SS. Previous work showed H. pylori strain SS1 biofilms expressed high amounts of the genes for the CagE ATPase, the CagL pilus protein, and the CagW protein of unknown function 16 . Consistent with these findings, biofilm proteome analysis identified upregulation of the CagA and CagD cag-T4SS proteins during biofilm formation 23 , or found that cagE is required for biofilm production 57 . While there is little overlap between the genes identified in these studies, the repeated overexpression or requirement for cag-T4SS genes in H. pylori biofilms suggest that the cag-T4SS plays an important role in this growth state. This idea is intriguing, as to date, the only known function of the cag-T4SS is in proinflammatory macromolecule delivery. Our data also showed that several genes of one of the other H. pylori T4SS’s were also upregulated in biofilm cells. This T4SS system, along with T4SS and ComB system, has been suggested to play a role in DNA uptake 58 . We found that many of the tfs4 system genes were upregulated, including virC1, virD4, virb11-like, HPG27_977, HPG27_972, HPG27_971, HPG27_969, and HPG27_968 (Supplementary Fig. 5 ). This extensive upregulation suggests these genes might be co-regulated by an as-yet-unknown regulator. The role of tfs4 during biofilm formation is not yet understood but it could increase DNA transfer between biofilm cells. This idea is supported by recent data from Bacillus subtilis, where biofilm formation drove high rates of conjugative ICE transfer compared to planktonic cells 59 . Such a situation might also increase the requirement for restriction-modification (R-M) systems, of which several were upregulated (Table 2 and Supplementary Table 2 ) or required for biofilm formation (Fig. 7 ). Taken together, our genetic and transcriptomic analyses suggest that H. pylori adjusts its phenotype when grown as biofilm, changing its metabolism to one that decreases central metabolism and benefits from alternate sources. H. pylori G27 can form a developed biofilm containing a mesh of flagella on gastric epithelial cells that resembles the one formed on abiotic surfaces. Flagella played a key structural role in both situations. In addition, we used genetic screens and transcriptomics to find new genes associated with H. pylori biofilm including genes encoding for acetone metabolism, hydrogen utilization, the cag T4SS and the relatively uncharacterized T4SS-4. Overall, it’s clear that the H. pylori biofilm is a growth state that H. pylori adopts under multiple conditions, requires a myriad set of abilities to create, and likely results in H. pylori with distinct physiology, including resistance to multiple antibiotics 20 , 21 , 22 . Methods Bacterial strain and growth conditions This study employed H. pylori wild-type and clinical strains, mutants generated using a transposon library that was constructed in strain G27 and generously provided by Nina Salama 60 , as well as mutants generated for this work (Table 4 ). Strains were grown on Columbia Horse Blood Agar (CHBA) (Difco), containing: 0.2% β-cyclodextrin, 10 μg of vancomycin per ml, 5 μg of cefsulodin per ml, 2.5 U of polymyxin B per ml, 5 μg of trimethoprim per ml, and 8 μg of amphotericin B per ml (all chemicals are from Thermo Fisher or Gold Biotech), or Brucella broth (Difco) containing 10% heat-inactivated fetal bovine serum (BB10; Gibco/BRL). Cultures were grown under microaerobic conditions (10% CO2, 5% O2, 85% N2) at 37 °C. For antibiotic resistance marker selection, bacterial media were supplemented with 25 μg of chloramphenicol (Cm) per ml or 75 μg kanamycin (Km) per ml. For growth curves, overnight cultures were diluted to OD 0.2 in fresh media and growth measurement were performed using an automatic microplate reader (Tecan Infinite F200), shaking at 3 g, until 24 h. OD600 readings were taken every 15 min with continuous shaking between readings. Table 4 Strains used for this in the present study. Biofilm formation on abiotic surfaces and crystal violet assay H. pylori strains were grown overnight with shaking in BB10, diluted to an OD600 of 0.15 with fresh BB10 media and 200 μl of the culture was used to fill triplicate wells of a sterile 96-well polystyrene microtiter plate (Costar, 3596). Wells at the edges of the microplate were filled with 200 μl of sterile water to avoid evaporation during incubation. Following static incubation of three days under microaerobic conditions, culture medium was removed by aspiration and the plate was washed twice using 1x phosphate-buffered saline (PBS). The wells were then filled with 200 μl of crystal violet (0.1%, wt/vol), and the plate was incubated for 2 min at room temperature. After removal of the crystal violet solution by aspiration, the plate was washed twice with PBS and air dried for 20 min at room temperature. To visualize biofilms, 200 μl of 70% ethanol (vol/vol) was added to the wells and the absorbance at 595 nm was measured. Confocal laser-scanning microscopy Biofilms of H. pylori G27 and mutants were prepared as described above using BB10 media, however, for confocal laser-scanning microscopy (CLSM), μ-Slide 8-well glass bottom chamber slides (ibidi, Germany) were used instead of 96-well microtiter plates. Three-day-old biofilms were stained with FM®1–43 (Invitrogen) or FilmTracer LIVE/DEAD biofilm viability kit (Invitrogen) according to the manufacturer’s instructions. Stained biofilms were visualized by CLSM with an LSM 5 Pascal laser-scanning microscope (Zeiss) and images were acquired using Imaris software (Bitplane). Biomass analysis of biofilm was carried out using FM®1–43 stained z-stack images (0.15 μm thickness) obtained by CLSM from randomly selected areas. The biomass of biofilms was determined using COMSTAT (27). Cell culture AGS (ATCC CRL 1739) human gastric epithelial cells were obtained directly from the American Type Culture Collection (ATCC) and maintained in RPMI-1640 medium (RPMI, Gibco) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco) at 37 °C under 10% CO2. For co-culture with H. pylori, AGS cells were maintained in DMEM/Ham’s F-12 medium (Gibco) with 10% FBS. AGS cell attachment and biofilm formation For assessment of AGS cell attachment and biofilm formation, H. pylori were maintained on CHBA, scrapped from the plate, resuspended in BB10 medium, and grown overnight with shaking. After an overnight growth, bacterial cells were harvested by centrifugation and resuspended in a pre-warmed mixture of DMEM/F-12 Medium with 10% FBS at a concentration of 1 × 108 CFU/ml. AGS cells were seeded in 24-well culture plates and cultured to reach 85–90% confluency. Number of cells were estimated using a hemocytometer. The AGS cell RPMI-1640 media was replaced by 1 ml fresh resuspended H. pylori culture at a multiplicity of infection (MOI) of 10. Attachment was assessed after a total of 1 h of coincubation. H. pylori was added without centrifugation and incubated at 37 °C under 10% CO2 for 1 h. However, since flagella and motility might play a role in surface colonization, we also tested H. pylori upon a 250 × g centrifugation for 30 min to force bacterial cells being in contact with AGS cells and then incubated at 37 °C under 10% CO2 for another 30 min. For biofilm formation, H. pylori was added without centrifugation and incubated at 37 °C under 10% CO2 for 3 days. The H. pylori-AGS cell culture was daily washed with 1 x PBS (twice) to remove unattached H. pylori cells. H. pylori-AGS cell culture was collected by trypsinization (trypsin/EDTA (Gibco)) and harvested in fresh DMEM/Ham’s F-12/FBS media. Bacterial sample was plated to determine the bacterial numbers. Scanning electron microscopy H. pylori wild-type G27 was grown on round glass coverslips in 6-well plates (12 mm, Costar) by dispersing 4 ml of a culture diluted to OD 0.15 in BB10. For biofilm formed on biotic surface, AGS cells were first maintained in DMEM/Ham’s F-12 medium with 10% FBS in 6-well plates containing glass coverslips. Once confluent, cells were washed twice with 1 x PBS and 1 × 108 CFU/ml of H. pylori wild-type G27 culture in mixture DMEM/F-12 Medium with 10% FBS was added to the wells. For both cultures, plates were incubated for 3-days under microaerobic conditions. Glass coverslips containing biofilm alone or AGS cells and H. pylori were washed twice with PBS and fixed with 2.5% glutaraldehyde (v/v) for 1 h at room temperature. Samples were then dehydrated with ethanol (10%, 25%, 50%, 75%, 90%, 100%), critical point dried (model: blazers Union 342/11 120B), sputtered with ~20 nm of gold (model: Technics Hummer VI) and imaged with a FEI Quanta 3D Dual beam SEM operating at 5 kV and 6.7 pA at the SEM facility at University of California Santa Cruz. Screening approach for biofilm-defective mutants A Tn-7-based H. pylori G27 transposon mutant library 43 was grown overnight in BB10 supplemented with 25 μg of chloramphenicol, diluted to an OD600 of 0.15 and then used to fill duplicate wells of a 6-well plate (Costar® 3516, Corning, Corning, NY, USA). After an hour of incubation, media containing non-attached, planktonic bacteria and/or potential biofilm-defective mutants was removed, and transferred to a new sterile 6-well plate and incubated again. The procedure was repeated at different incubation time (i.e., 2, 3, 24, 48, or 72 h). After 72 h, this enriched biofilm-defective sample was plated on CHBA media and individual colonies were isolated and stored at −80 °C. A total of 97 potential biofilm-defective mutants was isolated from this enrichment approach, and subsequently tested for abnormal biofilm formation using the crystal violet biofilm assay described above. Identification of transposon interrupted genes Nested PCR was used to identify the site of transposon insertions in biofilm-defective mutants, using methods described in Salama et al. 43 . For the first round of PCR, random primers with constant 5′ tail regions in tandem with a transposon-specific primer (Upstream or Downstream) were used (Supplementary Table 4 ). For the second round, a primer specific to the transposon was utilized with a primer complementary to the tail region of the original random primer. Therefore, the final products contain a portion of the transposon along with surrounding genomic information. These PCR products were sequenced (Sequetech, Mountain View, CA) and then compared to the G27 genomic sequence 49 at the UCSC Microbial Genome Browser (www. http://microbes.ucsc.edu ). Biofilm and planktonic growth conditions for transcriptomic analysis H. pylori G27 cells for transcriptomics were collected either from biofilm or from planktonic cultures under microaerobic conditions. In each case, cells were harvested by centrifugation (10 min, 1500 × g), washed two-times with ice cold PBS, and resuspended in 1 ml of Trizol Max (Ambion Bacterial Enhancement Kit, Ambion, Life technology, Carlsbad, CA, USA). For biofilm cells, H. pylori (4 ml per well) was grown in BB10 in 6-well plates (Costar), without shaking, for three days, and then scrapped from the surface with a sterile cell scraper. For planktonic cells, H. pylori was grown in BB10 in glass flasks with shaking for 24 h. Please note that the majority of both the planktonic (24 h) and biofilm (72 h) grown cultures are in the coccoid form. RNA extraction and library construction Total RNA from H. pylori in Trizol Max was extracted using the Trizol Max Bacterial Enhancement Kit (Ambion, Life Technology, Carlsbad, CA, USA) as described by the manufacturer. RNA was further purified and concentrated using an RNAeasy Kit (Qiagen). rRNA was removed using RiboZero magnetic kit (Illumina). Sequencing libraries were generated using NEBNext UltraTM Directional RNA library Prep Kit for Illumina (NEB, USA). Complementary DNA (cDNA) library quality and amount were verified using Agilent Bioanalyzer 2100 system (Agilent technologies, CA, USA) and then sequenced using Illumina NextSeq Mid-Output (UC Davis Genome Center). Transcriptomic analysis RNA-seq data were analyzed using CLC Genomics Workbench (version 11.0, CLC Bio, Boston, MA, USA). All sequences were trimmed, and forward and reverse sequenced reads generated for each growth state (biofilm vs. planktonic; three biological replicates for each condition) were mapped against the G27 reference genome 49 to quantify gene expression levels for each experimental condition. The expression value was measured in Reads per Kilobase Per Million Mapped Reads (RPKM). Genes were considered as differentially expressed when log2 (fold-change) was above 1 or below −1 and with statistical significance (P-value < 0.05, false-discovery rate (FDR < 0.001). Quantitative PCR To validate RNA-seq data, qRT-PCR was performed to quantify the transcription of six selected genes (we randomly selected two upregulated; virC1 and virB11, two downregulated; fdxB and HPG27_526 and two non-differentially expressed genes; lctP and hopC). Total RNA from three independent experiments was obtained and used for qRT-PCR. Primers were designed using Primer-Blast ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) and are listed in Supplementary Table 4 . The same amount of total RNA (1 μg/μl) was reverse transcribed using the LunaScriptTM RT SuperMix Kit (NEB) and qPCR reactions were prepared using Luna Universal qPCR Master Mix kit (NEB) The run was performed in ConnectTM Thermal Cycler (Bio-Rad) with the following cycling parameters: 2 min at 95 °C, followed by 39 cycle of 30 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. Relative expression of each gene was normalized to that of the 16S gene, whose expression was consistent throughout the different conditions. Quantitative measures were made using the 2−ΔΔCT method 61 . Three biological replicates and two technical replicates of each condition were performed. Design and construction of mutants Clean gene knockout constructs were designed to fully replace the target gene’s coding sequence with a chloramphenicol-resistance cassette (cat) or a kanamycin resistance cassette (aphA3), and synthesized by Twist Bioscience (San Francisco, CA). All constructs (Table 1 and Supplementary Table 1 ) were cloned in the pTwist Amp high copy vector and transformed into Escherichia coli DH10B. Purified vector containing the construct (2 to 10 μg) was used to transform H. pylori G27 WT. Mutants were selected on chloramphenicol or kanamycin-containing plates, colony purified, and confirmed by PCR using primers that flank antibiotic cassette (Supplementary Table 4 ). Statistical analysis Biofilm data were analyzed statistically using GraphPad Prism software (version 7, GraphPad Software Inc., San Diego, CA) by application of Wilcoxon–Mann–Whitney test or one-way ANOVA with Bartlett’s test. P < 0.05 or <0.01 were considered reflecting statistically significant. Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article. Data availability The data sets generated during and/or analyzed during the current study are either shown in the manuscript or available from the corresponding author on reasonable request. References 1. Uemura, N. et al. Helicobacter pylori infection and the development of gastric cancer. N. Engl. J. Med. 345, 784–789 (2001). 2. Parkin, D. M., Bray, F., Ferlay, J. & Pisani, P. Global cancer statistics, 2002. CA Cancer J. Clin. 55, 74–108 (2005). 3. Plummer, M., Franceschi, S., Vignat, J., Forman, D. & de Martel, C. Global burden of gastric cancer attributable to Helicobacter pylori. Int J. Cancer 136, 487–490 (2015). 4. Chey, W. D., Leontiadis, G. I., Howden, C. W. & Moss, S. F. ACG clinical guideline: treatment of Helicobacter pylori infection. Am. J. Gastroenterol. 112, 212–239 (2017). 5. Fallone, C. A. et al. 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Carron, M. A., Tran, V. R., Sugawa, C. & Coticchia, J. M. Identification of Helicobacter pylori biofilms in human gastric mucosa. J. Gastrointest. Surg. 10, 712–717 (2006). 19. Cammarota, G. et al. Biofilm demolition and antibiotic treatment to eradicate resistant Helicobacter pylori: a clinical trial. Clin. Gastroenterol. Hepatol. 8, 817–820 e813 (2010). 20. Yonezawa, H. et al. Impact of Helicobacter pylori biofilm formation on clarithromycin susceptibility and generation of resistance mutations. PLoS ONE 8, e73301 (2013). 21. Yonezawa, H., Osaki, T., Hojo, F. & Kamiya, S. Effect of Helicobacter pylori biofilm formation on susceptibility to amoxicillin, metronidazole and clarithromycin. Micro. Pathog. 132, 100–108 (2019). 22. Hathroubi, S., Zerebinski, J., Clarke, A. & Ottemann, K. M. Helicobacter pylori biofilm confers antibiotic tolerance in part via a protein-dependent mechanism. Antibiotics (Basel) 9, 355 (2020). 23. Shao, C. et al. Changes of proteome components of Helicobacter pylori biofilms induced by serum starvation. Mol. Med. Rep. 8, 1761–1766 (2013). 24. Wong, E. H. et al. Comparative genomics revealed multiple Helicobacter pylori genes associated with biofilm formation in Vitro. PLoS ONE 11, e0166835 (2016). 25. Hathroubi, S., Servetas, S. L., Windham, I., Merrell, D. S. & Ottemann, K. M. Helicobacter pylori biofilm formation and its potential role in pathogenesis. Microbiol. Mol. Biol. Rev. 82, e00001–e00018 (2018). 26. Windham, I. H. et al. Helicobacter pylori biofilm formation is differentially affected by common culture conditions, and proteins play a central role in the biofilm matrix. Appl. Environ. Microbiol. 84, e00391–18 (2018). 27. Grande, R. et al. Extracellular DNA in Helicobacter pylori biofilm: a backstairs rumour. J. Appl. Microbiol. 110, 490–498 (2011). 28. Servetas, S. L. et al. ArsRS-dependent regulation of homB contributes to Helicobacter pylori biofilm formation. Front. 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Diversification of the AlpB outer Membrane protein of Helicobacter pylori affects biofilm formation and cellular adhesion. J. Bacteriol. 199, e00729–16 (2017). 40. Kiedrowski, M. R. et al. Staphylococcus aureus biofilm growth on cystic fibrosis airway epithelial cells is enhanced during respiratory syncytial virus coinfection. mSphere 3, e00341–18 (2018). 41. Tharmalingam, N., Port, J., Castillo, D. & Mylonakis, E. Repurposing the anthelmintic drug niclosamide to combat Helicobacter pylori. Sci. Rep. 8, 3701 (2018). 42. Kim, N. et al. Genes of Helicobacter pylori regulated by attachment to AGS cells. Infect. Immun. 72, 2358–2368 (2004). 43. Salama, N. R., Shepherd, B. & Falkow, S. Global transposon mutagenesis and essential gene analysis of Helicobacter pylori. J. Bacteriol. 186, 7926–7935 (2004). 44. Brahmachary, P. et al. The human gastric pathogen Helicobacter pylori has a potential acetone carboxylase that enhances its ability to colonize mice. BMC Microbiol. 8, 14 (2008). 45. Ryan, K. A., Karim, N., Worku, M., Penn, C. W. & O’Toole, P. W. Helicobacter pylori flagellar hook-filament transition is controlled by a FliK functional homolog encoded by the gene HP0906. J. Bacteriol. 187, 5742–5750 (2005). 46. Radin, J. N. et al. Flagellar localization of a Helicobacter pylori autotransporter protein. MBio 4, e00613–00612 (2013). 47. Mittl, P. R., Luthy, L., Hunziker, P. & Grutter, M. G. The cysteine-rich protein A from Helicobacter pylori is a beta-lactamase. J. Biol. Chem. 275, 17693–17699 (2000). 48. Benoit, S., Mehta, N., Wang, G., Gatlin, M. & Maier, R. J. Requirement of hydD, hydE, hypC and hypE genes for hydrogenase activity in Helicobacter pylori. Micro. Pathog. 36, 153–157 (2004). 49. Baltrus, D. A. et al. The complete genome sequence of Helicobacter pylori strain G27. J. Bacteriol. 191, 447–448 (2009). 50. Rader, B. A. et al. Helicobacter pylori perceives the quorum-sensing molecule AI-2 as a chemorepellent via the chemoreceptor TlpB. 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Glycobiology 15, 721–733 (2005). 57. Cole, S. P., Harwood, J., Lee, R., She, R. & Guiney, D. G. Characterization of monospecies biofilm formation by Helicobacter pylori. J. Bacteriol. 186, 3124–3132 (2004). 58. Yuan, X. Y., Wang, Y. & Wang, M. Y. The type IV secretion system in Helicobacter pylori. Future Microbiol. 13, 1041–1054 (2018). 59. Lecuyer, F. et al. Biofilm formation drives transfer of the conjugative element ICEBs1 in Bacillus subtilis. mSphere 3, e00473–18 (2018). 60. Baldwin, D. N. et al. Identification of Helicobacter pylori genes that contribute to stomach colonization. Infect. Immun. 75, 1005–1016 (2007). 61. Rao, X., Huang, X., Zhou, Z. & Lin, X. An improvement of the 2^(−delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat. Bioinforma. Biomath. 3, 71–85 (2013). Acknowledgements This work was supported by National institute of Allergy and Infectious Diseases (NIAID) grant RO1AI116946 (to K.M.O), funds from the Santa Cruz Cancer Benefits Group (to K.M.O.) and NIH S10 grant 1S10OD023528 (to F. Yildiz). We would like to acknowledge Robert Maier (University of Georgia), Robin M. Delahay (University of Nottingham) and Fitnat Yildiz (University of California, Santa Cruz) for their helpful suggestions and comments on the study. We thank Nina Salama (Fred Hutchinson Cancer Center) for providing the transposon library; Ben Abrams (University of California, Santa Cruz) for confocal microscope training and assistance; Tom Yuvzinsky (University of California, Santa Cruz) for electron microscopy; the W. M. Keck Center for Nanoscale Optofluidics of University of California, Santa Cruz for use of the FEI Quanta 3D Dual beam microscope. Finally, we thank members of the Ottemann Lab for helpful discussions and comments on the manuscript. Author information Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ .

Robert Maier Investments

6 Investments

Robert Maier has made 6 investments. Their latest investment was in ekko as part of their Seed VC on January 1, 2022.

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Robert Maier Investments Activity

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Date

Round

Company

Amount

New?

Co-Investors

Sources

1/24/2022

Seed VC

ekko

Yes

1

10/20/2021

Seed VC

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$99M

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10

5/12/2021

Seed VC

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10

4/20/2021

Angel

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10

3/4/2019

Series C

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$99M

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10

Date

1/24/2022

10/20/2021

5/12/2021

4/20/2021

3/4/2019

Round

Seed VC

Seed VC

Seed VC

Angel

Series C

Company

ekko

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Amount

$99M

$99M

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Yes

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Co-Investors

Sources

1

10

10

10

10

Robert Maier Portfolio Exits

1 Portfolio Exit

Robert Maier has 1 portfolio exit. Their latest portfolio exit was Flaschenpost on November 01, 2020.

Date

Exit

Companies

Valuation
Valuations are submitted by companies, mined from state filings or news, provided by VentureSource, or based on a comparables valuation model.

Acquirer

Sources

11/1/2020

Acquired

$99M

1

Date

11/1/2020

Exit

Acquired

Companies

Valuation

$99M

Acquirer

Sources

1

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