A Potential Application of Pseudomonas psychrotolerans IALR632 for Lettuce Growth Promotion in Hydroponics.

Controlled environment agriculture hydroponic systems grow plants year-round without restriction from outside environmental conditions. In order to further improve crop yield, plant growth-promoting bacteria were tested on hydroponically grown lettuce (Lactuca sativa) plants. From our bacterial endophyte library, we found one bacterium, Pseudomonas psychrotolerans IALR632, that is promising in promoting lettuce growth in multiple hydroponic systems. When Green Oakleaf lettuce seeds were inoculated with IALR632 during germination, IALR632 significantly increased lateral root development by 164%. When germinated seedlings were inoculated with IALR632 and then transplanted to different hydroponic systems, shoot and root fresh weights of Green Oakleaf increased by 55.3% and 17.2% in a nutrient film technique (NFT) system in the greenhouse, 13.5% and 13.8% in an indoor vertical NFT system, and 15.3% and 13.6% in a deep water cultivation (DWC) system, respectively. IALR632 also significantly increased shoot fresh weights of Rex by 33.9%, Red Oakleaf by 21.0%, Red Sweet Crisp by 15.2%, and Nancy by 29.9%, as well as Red Rosie by 8.6% (no significant difference). Inoculation of IALR632-GFP and subsequent analysis by confocal microscopy demonstrated the endophytic nature and translocation from roots to shoots. The results indicate that P. psychrotolerans IALR632 has a potential application in hydroponically grown lettuce plants.

SAMHD1 Regulates Human Papillomavirus 16-Induced Cell Proliferation and Viral Replication during Differentiation of Keratinocytes.

Human papillomaviruses induce a host of anogenital cancers, as well as oropharyngeal cancer (HPV+OPC); human papillomavirus 16 (HPV16) is causative in around 90% of HPV+OPC cases. Using telomerase reverse transcriptase (TERT) immortalized foreskin keratinocytes (N/Tert-1), we have identified significant host gene reprogramming by HPV16 (N/Tert-1+HPV16) and demonstrated that N/Tert-1+HPV16 support late stages of the viral life cycle. Expression of the cellular dNTPase and homologous recombination factor sterile alpha motif and histidine-aspartic domain HD-containing protein 1 (SAMHD1) is transcriptionally regulated by HPV16 in N/Tert-1. CRISPR/Cas9 removal of SAMHD1 from N/Tert-1 and N/Tert-1+HPV16 demonstrates that SAMHD1 controls cell proliferation of N/Tert-1 only in the presence of HPV16; the deletion of SAMHD1 promotes hyperproliferation of N/Tert-1+HPV16 cells in organotypic raft cultures but has no effect on N/Tert-1. Viral replication is also elevated in the absence of SAMHD1. This new system has allowed us to identify a specific interaction between SAMHD1 and HPV16 that regulates host cell proliferation and viral replication; such studies are problematic in nonimmortalized primary keratinocytes due to their limited life span. To confirm the relevance of our results, we repeated the analysis with human tonsil keratinocytes (HTK) immortalized by HPV16 (HTK+HPV16) and observed the same hyperproliferative phenotype following CRISPR/Cas9 editing of SAMHD1. Identical results were obtained with three independent CRISPR/Cas9 guide RNAs. The isogenic pairing of N/Tert-1 with N/Tert-1+HPV16, combined with HTK+HPV16, presents a unique system to identify host genes whose products functionally interact with HPV16 to regulate host cellular growth in keratinocytes.IMPORTANCE HPVs are causative agents in human cancers and are responsible for around of 5% of all cancers. A better understanding of the viral life cycle in keratinocytes will facilitate the development of novel therapeutics to combat HPV-positive cancers. Here, we present a unique keratinocyte model to identify host proteins that specifically interact with HPV16. Using this system, we report that a cellular gene, SAMHD1, is regulated by HPV16 at the RNA and protein levels in keratinocytes. Elimination of SAMHD1 from these cells using CRISPR/Cas9 editing promotes enhanced cellular proliferation by HPV16 in keratinocytes and elevated viral replication but not in keratinocytes that do not have HPV16. Our study demonstrates a specific intricate interplay between HPV16 and SAMHD1 during the viral life cycle and establishes a unique model system to assist exploring host factors critical for HPV pathogenesis.

Human Papillomavirus 16 E2 Regulates Keratinocyte Gene Expression Relevant to Cancer and the Viral Life Cycle.

Human papillomaviruses (HPVs) are causative agents in ano-genital and oropharyngeal cancers. The virus must reprogram host gene expression to promote infection, and E6 and E7 contribute to this via the targeting of cellular transcription factors, including p53 and pRb, respectively. The HPV16 E2 protein regulates host gene expression in U2OS cells, and in this study, we extend these observations into telomerase reverse transcriptase (TERT) immortalized oral keratinocytes (NOKs) that are capable of supporting late stages of the HPV16 life cycle. We observed repression of innate immune genes by E2 that are also repressed by the intact HPV16 genome in NOKs. Transcriptome sequencing (RNA-seq) data identified 167 up- and 395 downregulated genes by E2; there was a highly significant overlap of the E2-regulated genes with those regulated by the intact HPV16 genome in the same cell type. Small interfering RNA (siRNA) targeting of E2 reversed the repression of E2-targeted genes. The ability of E2 to repress innate immune genes was confirmed in an ano-genital immortalized keratinocyte cell line, N/Tert-1. We present the analysis of data from The Cancer Genome Atlas (TCGA) for HPV16-positive and -negative head and neck cancers (HNC) suggesting that E2 plays a role in the regulation of the host genome in cancers. Patients with HPV16-positive HNC with a loss of E2 expression exhibited a worse clinical outcome, and we discuss how this could, at least partially, be related to the loss of E2 host gene regulation.IMPORTANCE Human papillomavirus 16 (HPV16)-positive tumors that retain expression of E2 have a better clinical outcome than those that have lost E2 expression. It has been suggested that this is due to a loss of E2 repression of E6 and E7 expression, but this is not supported by data from tumors where there is not more E6 and E7 expression in the absence of E2. Here we report that E2 regulates host gene expression and place this regulation in the context of the HPV16 life cycle and HPV16-positive head and neck cancers (the majority of which retain E2 expression). We propose that this E2 function may play an important part in the increased response of HPV16-positive cancers to radiation therapy. Therefore, host gene regulation by E2 may be important for promotion of the HPV16 life cycle and also for the response of HPV16-positive tumors to radiation therapy.

An oral keratinocyte life cycle model identifies novel host genome regulation by human papillomavirus 16 relevant to HPV positive head and neck cancer.

Many aspects of the HPV life cycle have been characterized in cervical cell lines (W12, CIN612) and in HPV immortalized primary foreskin keratinocytes. There is now an epidemic of HPV positive oropharyngeal cancers (HPV16 is responsible for 80-90% of these); therefore increased understanding of the HPV16 life cycle in oral keratinocytes is a priority. To date there have been limited reports characterizing the HPV16 life cycle in oral keratinocytes. Using TERT immortalized "normal" oral keratinocytes (NOKs) we generated clonal cell lines maintaining the HPV16 genome as an episome, NOKs+HPV16. Organotypic raft cultures demonstrated appropriate expression of differentiation markers, E1^E4 and E2 expression along with amplification of the viral genome in the upper layers of the epithelium. Using this unique system RNA-seq analysis revealed extensive gene regulation of the host genome by HPV16; many of the changes have not been observed for HPV16 before. The RNA-seq data was validated on a key set of anti-viral innate immune response genes repressed by HPV16 in NOKs+HPV16. We show that the behavior of these NOKs+HPV16 lines is identical to HPV16 immortalized human tonsil keratinocytes with regards innate gene regulation. Finally, using The Cancer Genome Atlas (TCGA) data we examined gene expression patterns from HPV positive and negative head and neck cancers and demonstrate this innate immune gene signature set is also downregulated in HPV positive cancers versus negative. Our system provides a model for understanding HPV16 transcriptional regulation of oral keratinocytes that is directly relevant to HPV positive head and neck cancer.

DNA Damage Reduces the Quality, but Not the Quantity of Human Papillomavirus 16 E1 and E2 DNA Replication.

Human papillomaviruses (HPVs) are causative agents in almost all cervical carcinomas. HPVs are also causative agents in head and neck cancer, the cases of which are increasing rapidly. Viral replication activates the DNA damage response (DDR) pathway; associated proteins are recruited to replication foci, and this pathway may serve to allow for viral genome amplification. Likewise, HPV genome double-strand breaks (DSBs) could be produced during replication and could lead to linearization and viral integration. Many studies have shown that viral integration into the host genome results in unregulated expression of the viral oncogenes, E6 and E7, promoting HPV-induced carcinogenesis. Previously, we have demonstrated that DNA-damaging agents, such as etoposide, or knocking down viral replication partner proteins, such as topoisomerase II ß binding protein I (TopBP1), does not reduce the level of DNA replication. Here, we investigated whether these treatments alter the quality of DNA replication by HPV16 E1 and E2. We confirm that knockdown of TopBP1 or treatment with etoposide does not reduce total levels of E1/E2-mediated DNA replication; however, the quality of replication is significantly reduced. The results demonstrate that E1 and E2 continue to replicate under genomically-stressed conditions and that this replication is mutagenic. This mutagenesis would promote the formation of substrates for integration of the viral genome into that of the host, a hallmark of cervical cancer.

Growth of Escherichia coli O157:H7, Non-O157 Shiga Toxin-Producing Escherichia coli , and Salmonella in Water and Hydroponic Fertilizer Solutions.

The desire for local, fresh produce year round is driving the growth of hydroponic growing systems in the United States. Many food crops, such as leafy greens and culinary herbs, grown within hydroponics systems have their root systems submerged in recirculating nutrient-dense fertilizer solutions from planting through harvest. If a foodborne pathogen were introduced into this water system, the risk of contamination to the entire crop would be high. Hence, this study was designed to determine whether Escherichia coli O157:H7, non-O157 Shiga toxin-producing E. coli , and Salmonella were able to survive and reproduce in two common hydroponic fertilizer solutions and in water or whether the bacteria would be killed or suppressed by the fertilizer solutions. All the pathogens grew by 1 to 6 log CFU/ml over a 24-h period, depending on the solution. E. coli O157:H7 reached higher levels in the fertilizer solution with plants (3.12 log CFU/ml), whereas non-O157 Shiga toxin-producing E. coli and Salmonella reached higher levels in the fertilizer solution without plants (1.36 to 3.77 log CFU/ml). The foodborne pathogens evaluated here survived for 24 h in the fertilizer solution, and populations grew more rapidly in these solutions than in plain water. Therefore, human pathogens entering the fertilizer solution tanks in hydroponic systems would be expected to rapidly propagate and spread throughout the system and potentially contaminate the entire crop.

Presence of calcium-binding motifs in PilY1 homologs correlates with Ca-mediated twitching motility and evolutionary history across diverse bacteria.

Twitching motility, involving type IV pili, is essential for host colonization and virulence of many pathogenic bacteria. Studies of PilY1, a tip-associated type IV pili protein, indicate that PilY1 functions as a switch between pilus extension and retraction, resulting in twitching motility. Recent work detected a calcium-binding motif in PilY1 of some animal bacterial pathogens and demonstrated that binding of calcium to PilY1 with this motif regulates twitching. Though studies of PilY1 in non-animal pathogens are limited, our group demonstrated that twitching motility in the plant pathogen Xylella fastidiosa, which contains three PilY1 homologs, is increased by calcium supplementation. A study was conducted to investigate the phylogenetic relationship between multiple PilY1 homologs, the presence of calcium-binding motifs therein, and calcium-mediated twitching motility across diverse bacteria. Strains analyzed contained one to three PilY1 homologs, but phylogenetic analyses indicated that PilY1 homologs containing the calcium-binding motif Dx[DN]xDGxxD are phylogenetically divergent from other PilY1 homologs. Plant-associated bacteria included in these analyses were then examined for a calcium-mediated twitching response. Results indicate that bacteria must have at least one PilY1 homolog containing the Dx[DN]xDGxxD motif to display a calcium-mediated increase in twitching motility, which likely reflects an adaption to environmental calcium concentrations.