Engineering Phage Host-Range and Suppressing Bacterial Resistance through Phage Tail Fiber Mutagenesis.

The rapid emergence of antibiotic-resistant infections is prompting increased interest in phage-based antimicrobials. However, acquisition of resistance by bacteria is a major issue in the successful development of phage therapies. Through natural evolution and structural modeling, we identified host-range-determining regions (HRDRs) in the T3 phage tail fiber protein and developed a high-throughput strategy to genetically engineer these regions through site-directed mutagenesis. Inspired by antibody specificity engineering, this approach generates deep functional diversity while minimizing disruptions to the overall tail fiber structure, resulting in synthetic "phagebodies." We showed that mutating HRDRs yields phagebodies with altered host-ranges, and select phagebodies enable long-term suppression of bacterial growth in vitro, by preventing resistance appearance, and are functional in vivo using a murine model. We anticipate that this approach may facilitate the creation of next-generation antimicrobials that slow resistance development and could be extended to other viral scaffolds for a broad range of applications.

The global transcriptomes of Salmonella enterica serovars Gallinarum, Dublin and Enteritidis in the avian host.

Salmonella enterica serovar Gallinarum causes Fowl Typhoid in poultry, and it is host specific to avian species. The reasons why S. Gallinarum is restricted to avians, and at the same time predominately cause systemic infections in these hosts, are unknown. In the current study, we developed a surgical approach to study gene expression inside the peritoneal cavity of hens to shed light on this. Strains of the host specific S. Gallinarum, the cattle-adapted S. Dublin and the broad host range serovar, S. Enteritidis, were enclosed in semi-permeable tubes and surgically placed for 4 h in the peritoneal cavity of hens and for control in a minimal medium at 41.2 °C. Global gene-expression under these conditions was compared between serovars using tiled-micro arrays with probes representing the genome of S. Typhimurium, S. Dublin and S. Gallinarum. Among other genes, genes of SPI-13, SPI-14 and the macrophage survival gene mig-14 were specifically up-regulated in the host specific serovar, S. Gallinarum, and further studies into the role of these genes in host specific infection are highly indicated. Analysis of pathways and GO-terms, which were enriched in the host specific S. Gallinarum without being enriched in the two other serovars indicated that host specificity was characterized by a metabolic fine-tuning as well as unique expression of virulence associated pathways. The cattle adapted serovar S. Dublin differed from the two other serovars by a lack of up-regulation of genes encoded in the virulence associated pathogenicity island 2, and this may explain the inability of this serovar to cause disease in poultry.

Bacteriophage crosstalk: coordination of prophage induction by trans-acting antirepressors.

Many species of bacteria harbor multiple prophages in their genomes. Prophages often carry genes that confer a selective advantage to the bacterium, typically during host colonization. Prophages can convert to infectious viruses through a process known as induction, which is relevant to the spread of bacterial virulence genes. The paradigm of prophage induction, as set by the phage Lambda model, sees the process initiated by the RecA-stimulated self-proteolysis of the phage repressor. Here we show that a large family of lambdoid prophages found in Salmonella genomes employs an alternative induction strategy. The repressors of these phages are not cleaved upon induction; rather, they are inactivated by the binding of small antirepressor proteins. Formation of the complex causes the repressor to dissociate from DNA. The antirepressor genes lie outside the immunity region and are under direct control of the LexA repressor, thus plugging prophage induction directly into the SOS response. GfoA and GfhA, the antirepressors of Salmonella prophages Gifsy-1 and Gifsy-3, each target both of these phages' repressors, GfoR and GfhR, even though the latter proteins recognize different operator sites and the two phages are heteroimmune. In contrast, the Gifsy-2 phage repressor, GtgR, is insensitive to GfoA and GfhA, but is inactivated by an antirepressor from the unrelated Fels-1 prophage (FsoA). This response is all the more surprising as FsoA is under the control of the Fels-1 repressor, not LexA, and plays no apparent role in Fels-1 induction, which occurs via a Lambda CI-like repressor cleavage mechanism. The ability of antirepressors to recognize non-cognate repressors allows coordination of induction of multiple prophages in polylysogenic strains. Identification of non-cleavable gfoR/gtgR homologues in a large variety of bacterial genomes (including most Escherichia coli genomes in the DNA database) suggests that antirepression-mediated induction is far more common than previously recognized.

Cryo-EM analysis of Pseudomonas phage Pa193 structural components.

The World Health Organization has designated Pseudomonas aeruginosa as a critical pathogen for the development of new antimicrobials. Bacterial viruses, or bacteriophages, have been used in various clinical settings, commonly called phage therapy, to address this growing public health crisis. Here, we describe a high-resolution structural atlas of a therapeutic, contractile-tailed Pseudomonas phage, Pa193. We used bioinformatics, proteomics, and cryogenic electron microscopy single particle analysis to identify, annotate, and build atomic models for 21 distinct structural polypeptide chains forming the icosahedral capsid, neck, contractile tail, and baseplate. We identified a putative scaffolding protein stabilizing the interior of the capsid 5-fold vertex. We also visualized a large portion of Pa193 ~ 500 Å long tail fibers and resolved the interface between the baseplate and tail fibers. The work presented here provides a framework to support a better understanding of phages as biomedicines for phage therapy and inform engineering opportunities.

Engineering the Modular Receptor-Binding Proteins of Klebsiella Phages Switches Their Capsule Serotype Specificity.

The high specificity of bacteriophages is driven by their receptor-binding proteins (RBPs). Many Klebsiella bacteriophages target the capsular exopolysaccharide as the receptor and encode RBPs with depolymerase activity. The modular structure of these RBPs with an N-terminal structural module to attach the RBP to the phage tail, and a C-terminal specificity module for exopolysaccharide degradation, supports horizontal transfer as a major evolutionary driver for Klebsiella phage RBPs. We mimicked this natural evolutionary process by the construction of modular RBP chimeras, exchanging N-terminal structural modules and C-terminal specificity modules. All chimeras strictly follow the capsular serotype specificity of the C-terminal module. Transplanting chimeras with a K11 N-terminal structural RBP module in a Klebsiella phage K11 scaffold results in a capsular serotype switch and corresponding host range modification of the synthetic phages, demonstrating that horizontal transfer of C-terminal specificity modules offers Klebsiella phages an evolutionary highway for rapid adaptation to new capsular serotypes.IMPORTANCE The antimicrobial resistance crisis has rekindled interest in bacteriophage therapy. Phages have been studied over a century as therapeutics to treat bacterial infections, but one of the biggest challenges for the use of phages in therapeutic interventions remains their high specificity. In particular, many Klebsiella phages have a narrow spectrum constrained by the high diversity of exopolysaccharide capsules that shield access to the cells. In this work, we have elaborated how Klebsiella phages deal with this high diversity by exchanging building blocks of their receptor-binding proteins.

Polyamine depletion has global effects on stress and virulence gene expression and affects HilA translation in Salmonella enterica serovar typhimurium.

Polyamines are small cationic amines required for modulating multiple cell process, including cell growth and DNA and RNA stability. In Salmonella polyamines are primarily synthesized from L-arginine or L-ornithine. Based on a previous study, which demonstrated that polyamines affect the expression of virulence gene in S. Typhimurium, we investigated the role of polyamines in the global gene and protein expression in S. Typhimurium. The depletion of polyamine biosynthesis led to down-regulation of genes encoding structural components of the Type Three Secretion system 1 (TTSS1) and its secreted effectors. Interestingly, Expression of HilA, which is the master regulator of Salmonella Pathogenicity Island 1 (SPI1), was only reduced at the post-transcriptional in the polyamine mutant. Enzymes related to biosynthesis and/or transport of several amino acids were up-regulated, just as the Mg2+-transport systems were three to six-fold up-regulated at both the transcriptional and protein levels. Furthermore, in the polyamine depletion mutant, proteins related to stress response (IbpA, Dps, SodB), were 2-5 fold up-regulated. Together our data provide strong evidence that polyamine depletion affects expression of proteins linked with virulence and stress response of S. Typhimurium. Furthermore, polyamines positively affected translation of HilA, the major regulator of SPI1.

Post-transcriptional control of Crp-cAMP by RNase LS in Escherichia coli.

Escherichia coli ribonuclease LS was first characterized as a potential antagonist of bacteriophage T4; the E. coli rnlA gene is required for this activity. When rnlA mutant cells were grown on Luria-Bertani agar containing a high concentration of NaCl, their growth was substantially impaired, and introduction of a mutation into crp or cyaA alleviated the NaCl sensitivity. A mutation in rnlA caused fivefold overexpression of Crp. At the same time, the expression of sigma(38) was lower by two- to threefold in an rnlA mutant than in the wild type, which probably accounts for the susceptibility to high NaCl concentration. The overproduction of Crp was eliminated by deletion of the Crp-site II, to which Crp binds to enhance its own transcription in the presence of abnormally high concentration of cAMP. Consistently, introduction of a mutation into cyaA also eliminated the overproduction of Crp. In fact, all of CyaA, cAMP and cyaA transcripts accumulated to high levels and, after induction, cyaA transcripts were markedly stabilized in an rnlA mutant compared with the wild type. We conclude that RNase LS regulates Crp-cAMP concentration by degrading the cyaA transcripts.

The SPI-19 encoded type-six secretion-systems (T6SS) of Salmonella enterica serovars Gallinarum and Dublin play different roles during infection.

Salmonella Pathogenicity Islands 19 (SPI19) encodes a type VI secretion system (T6SS). SPI19 is only present in few serovars of S. enterica, including the host-adapted serovar S. Dublin and the host-specific serovar S. Gallinarum. The role of the SPI19 encoded T6SS in virulence in these serovar is not fully understood. Here we show that during infection of mice, a SPI19/T6SS deleted strain of S. Dublin 2229 was less virulent than the wild type strain after oral challenge, but not after IP challenge. The mutant strain also competed significantly poorer than the wild type strain when co-cultured with strains of E. coli, suggesting that this T6SS plays a role in pathogenicity by killing competing bacteria in the intestine. No significant difference was found between wild type S. Gallinarum G9 and its ΔSPI19/T6SS mutant in infection, whether chicken were challenged orally or by the IP route, and the S. Gallinarum G9 ΔSPI19/T6SS strain competed equally well as the wild type strain against strains of E. coli. However, contrary to what was observed with S. Dublin, the wild type G9 strains was significantly more cytotoxic to monocyte derived primary macrophages from hens than the mutant, suggesting that SPI19/T6SS in S. Gallinarum mediates killing of eukaryotic cells. The lack of significant importance of SPI19/T6SS after oral and systemic challenge of chicken was confirmed by knocking out SPI19 in a second strain, J91. Together the results suggest that the T6SS encoded from SPI19 have different roles in the two serovars and that it is a virulence-factor after oral challenge of mice in S. Dublin, while we cannot confirm previous results that SPI19/T6SS influence virulence significantly in S. Gallinarum.

A singular case of prophage complementation in mutational activation of recET orthologs in Salmonella enterica serovar Typhimurium.

A class of mutations that suppress the recombination defects of recB mutants in Salmonella enterica serovar Typhimurium strain LT2 activates the normally silent recET module of the Gifsy-1 prophage. Allele sbcE21 is a 794-bp deletion within the immunity region of the prophage. Concomitant with activating recET, sbcE21 stimulates Gifsy-1 excision, resulting in unstable suppression. Early studies found both recB suppression and its instability to depend on the presence of the related Gifsy-2 prophage elsewhere in the chromosome. In cells lacking Gifsy-2, the sbcE21 allele became stable but no longer corrected recB defects. Here, we show that a single Gifsy-2 gene is required for Gifsy-1 recET activation in the sbcE21 background. This gene encodes GtgR, the Gifsy-2 repressor. Significantly, the sbcE21 deletion has one end point within the corresponding gene in the Gifsy-1 genome, gogR, which in strain LT2 is a perfect duplicate of gtgR. The deletion truncates gogR and places the Gifsy-1 left operon, including the recET and xis genes, under the control of the gogR promoter. The ability of GtgR to trans-activate this promoter therefore implies that GtgR and GogR normally activate the transcription of their own genes. Consistent with the symmetry of the system, a similar deletion in Gifsy-2 results in a Gifsy-1-dependent sbc phenotype (sbcF24). Two additional Gifsy-1 deletions (sbcE23 and sbcE25) were characterized, as well. The latter causes all but the last codon of the gogR gene to fuse, in frame, to the second half of recE. The resulting hybrid protein appears to function as both a transcriptional regulator and a recombination enzyme.

Phage-Based Applications in Synthetic Biology.

Bacteriophage research has been instrumental to advancing many fields of biology, such as genetics, molecular biology, and synthetic biology. Many phage-derived technologies have been adapted for building gene circuits to program biological systems. Phages also exhibit significant medical potential as antibacterial agents and bacterial diagnostics due to their extreme specificity for their host, and our growing ability to engineer them further enhances this potential. Phages have also been used as scaffolds for genetically programmable biomaterials that have highly tunable properties. Furthermore, phages are central to powerful directed evolution platforms, which are being leveraged to enhance existing biological functions and even produce new ones. In this review, we discuss recent examples of how phage research is influencing these next-generation biotechnologies.

Genetic and molecular analysis of GogB, a phage-encoded type III-secreted substrate in Salmonella enterica serovar typhimurium with autonomous expression from its associated phage.

Salmonella enterica serovar Typhimurium is lysogenized by several temperate bacteriophages that encode lysogenic conversion genes, which can act as virulence factors during infection and contribute to the genetic diversity and pathogenic potential of the lysogen. We have investigated the temperate bacteriophage called Gifsy-1 in S.enterica serovar Typhimurium and show here that the product of the gogB gene encoded within this phage shares similarity with proteins from other Gram-negative pathogens. The amino-terminal portion of GogB shares similarity with leucine-rich repeat-containing virulence-associated proteins from other Gram-negative pathogens, whereas the carboxyl-terminal portion of GogB shares similarity with uncharacterized proteins in other pathogens. We show that GogB is secreted by both type III secretion systems encoded in Salmonella Pathogenicity Island-1 (SPI-1) and SPI-2 but translocation into host cells is a SPI-2-mediated process. Once translocated, GogB localizes to the cytoplasm of infected host cells. The genetic regulation of gogB in Salmonella is influenced by the transcriptional activator, SsrB, under SPI-2-inducing conditions, but the modular nature of the gogB gene allows for autonomous expression and type III secretion following horizontal gene transfer into a heterologous pathogen. These data define the first autonomously expressed lysogenic conversion gene within Gifsy-1 that acts as a modular and promiscuous type III-secreted substrate of the infection process.

Engineering Modular Viral Scaffolds for Targeted Bacterial Population Editing.

Bacteria are central to human health and disease, but existing tools to edit microbial consortia are limited. For example, broad-spectrum antibiotics are unable to accurately manipulate bacterial communities. Bacteriophages can provide highly specific targeting of bacteria, but assembling well-defined phage cocktails solely with natural phages can be a time-, labor- and cost-intensive process. Here, we present a synthetic-biology strategy to modulate phage host ranges by engineering phage genomes in Saccharomyces cerevisiae. We used this technology to redirect Escherichia coli phage scaffolds to target pathogenic Yersinia and Klebsiella bacteria, and conversely, Klebsiella phage scaffolds to target E. coli by modular swapping of phage tail components. The synthetic phages achieved efficient killing of their new target bacteria and were used to selectively remove bacteria from multi-species bacterial communities with cocktails based on common viral scaffolds. We envision that this approach will accelerate phage-biology studies and enable new technologies for bacterial population editing.

Removal of the phage-shock protein PspB causes reduction of virulence in Salmonella enterica serovar Typhimurium independently of NRAMP1.

The phage-shock protein (Psp) system is believed to manage membrane stress in all Enterobacteriaceae and has recently emerged as being important for virulence in several pathogenic species of this phylum. The core of the Psp system consists of the pspA-D operon and the distantly located pspG gene. In Salmonella enterica serovar Typhimurium (S. Typhimurium), it has recently been reported that PspA is essential for systemic infection of mice, but only in NRAMP1(+) mice, signifying that attenuation is related to coping with divalent cation starvation in the intracellular environment. In the present study, we investigated the contribution of individual psp genes to virulence of S. Typhimurium. Interestingly, deletion of the whole pspA-D set of genes caused attenuation in both NRAMP1(+) and NRAMP1(-) mice, indicating that one or more of the psp genes contribute to virulence independently of NRAMP1 expression in the host. Investigations of single gene mutants showed that knock out of pspB reduced virulence in both types of mice, while deletion of pspA only caused attenuation in NRAMP1(+) mice, and deletion of pspD had a minor effect in NRAMP1(-) mice, while deletions of either pspC or pspG did not affect virulence. Experiments addressed at elucidating the role of PspB in virulence revealed that PspB is dispensable for uptake to and intracellular replication in cultured macrophages and resistance to complement-induced killing. Furthermore, the Psp system of S. Typhimurium was dispensable during pIV-induced secretin stress. In conclusion, our results demonstrate that removal of PspB reduces virulence in S. Typhimurium independently of host NRAMP1 expression, demonstrating that PspB has roles in intra-host survival distinct from the reported contributions of PspA.

Identification of metabolic pathways essential for fitness of Salmonella Typhimurium in vivo.

Bacterial infections remain a threat to human and animal health worldwide, and there is an urgent need to find novel targets for intervention. In the current study we used a computer model of the metabolic network of Salmonella enterica serovar Typhimurium and identified pairs of reactions (cut sets) predicted to be required for growth in vivo. We termed such cut sets synthetic auxotrophic pairs. We tested whether these would reveal possible combined targets for new antibiotics by analyzing the performance of selected single and double mutants in systemic mouse infections. One hundred and two cut sets were identified. Sixty-three of these included only pathways encoded by fully annotated genes, and from this sub-set we selected five cut sets involved in amino acid or polyamine biosynthesis. One cut set (asnA/asnB) demonstrated redundancy in vitro and in vivo and showed that asparagine is essential for S. Typhimurium during infection. trpB/trpA as well as single mutants were attenuated for growth in vitro, while only the double mutant was a cut set in vivo, underlining previous observations that tryptophan is essential for successful outcome of infection. speB/speF,speC was not affected in vitro but was attenuated during infection showing that polyamines are essential for virulence apparently in a growth independent manner. The serA/glyA cut-set was found to be growth attenuated as predicted by the model. However, not only the double mutant, but also the glyA mutant, were found to be attenuated for virulence. This adds glycine production or conversion of glycine to THF to the list of essential reactions during infection. One pair (thrC/kbl) showed true redundancy in vitro but not in vivo demonstrating that threonine is available to the bacterium during infection. These data add to the existing knowledge of available nutrients in the intra-host environment, and have identified possible new targets for antibiotics.

The putative thiosulfate sulfurtransferases PspE and GlpE contribute to virulence of Salmonella Typhimurium in the mouse model of systemic disease.

The phage-shock protein PspE and GlpE of the glycerol 3-phosphate regulon of Salmonella enterica serovar Typhimurium are predicted to belong to the class of thiosulfate sulfurtransferases, enzymes that traffic sulfur between molecules. In the present study we demonstrated that the two genes contribute to S. Typhimurium virulence, as a glpE and pspE double deletion strain showed significantly decreased virulence in a mouse model of systemic infection. However, challenge of cultured epithelial cells and macrophages did not reveal any virulence-associated phenotypes. We hypothesized that their contribution to virulence could be in sulfur metabolism or by contributing to resistance to nitric oxide, oxidative stress, or cyanide detoxification. In vitro studies demonstrated that glpE but not pspE was important for resistance to H2O2. Since the double mutant, which was the one affected in virulence, was not affected in this assay, we concluded that resistance to oxidative stress and the virulence phenotype was most likely not linked. The two genes did not contribute to nitric oxid stress, to synthesis of essential sulfur containing amino acids, nor to detoxification of cyanide. Currently, the precise mechanism by which they contribute to virulence remains elusive.

Escherichia coli rnlA and rnlB compose a novel toxin-antitoxin system.

RNase LS was originally identified as a potential antagonist of bacteriophage T4 infection. When T4 dmd is defective, RNase LS activity rapidly increases after T4 infection and cleaves T4 mRNAs to antagonize T4 reproduction. Here we show that rnlA, a structural gene of RNase LS, encodes a novel toxin, and that rnlB (formally yfjO), located immediately downstream of rnlA, encodes an antitoxin against RnlA. Ectopic expression of RnlA caused inhibition of cell growth and rapid degradation of mRNAs in ΔrnlAB cells. On the other hand, RnlB neutralized these RnlA effects. Furthermore, overexpression of RnlB in wild-type cells could completely suppress the growth defect of a T4 dmd mutant, that is, excess RnlB inhibited RNase LS activity. Pull-down analysis showed a specific interaction between RnlA and RnlB. Compared to RnlA, RnlB was extremely unstable, being degraded by ClpXP and Lon proteases, and this instability may increase RNase LS activity after T4 infection. All of these results suggested that rnlA-rnlB define a new toxin-antitoxin (TA) system.

Loss of Hfq activates the sigmaE-dependent envelope stress response in Salmonella enterica.

Ubiquitous RNA-binding protein Hfq mediates the regulatory activity of many small RNAs (sRNAs) in bacteria. To identify potential targets for Hfq-mediated regulation in Salmonella, we searched for lacZ translational fusions whose activity varied in the presence or absence of Hfq. Fusions downregulated by Hfq were more common than fusions showing the opposite response. Surprisingly, in a subset of isolates from the major class, the higher activity in the absence of Hfq was due to transcriptional activation by the alternative sigma factor RpoE (sigmaE). Activation of the sigmaE regulon normally results from envelope stress conditions that elicit proteolytic cleavage of the anti-sigmaE factor RseA. Using an epitope tagged variant of RseA, we found that RseA is cleaved at an increased rate in a strain lacking Hfq. This cleavage was dependent on the DegS protease and could be completely prevented upon expressing the hfq gene from an inducible promoter. Thus, loss of Hfq function appears to affect envelope biogenesis in a way that mimics a stress condition and thereby induces the sigmaE response constitutively. In a RseA mutant, activation of the sigmaE response causes Hfq-dependent downregulation of outer membrane protein (OMP) genes including lamB, ompA, ompC and ompF. For ompA, downregulation results in part from sigmaE-dependent accumulation of MicA (SraD), a small RNA recently shown to downregulate ompA transcript levels in stationary phase. We show that the micA gene is under sigmaE control, and that DegS-mediated sigmaE release is required for the accumulation of MicA RNA upon entry into stationary phase. A similar mechanism involving additional, still unidentified, sRNAs, might underlie the growth phase-dependent regulation of other OMP mRNAs.