Search

Coliphage lambda proteins Rz and Rz1 are the inner membrane and outer membrane subunits of the spanin complex—a heterotetramer that bridges the periplasm and is essential for the disruption of the outer membrane during phage lysis. Recent evidence suggests the spanin complex functions by fusing the inner and outer membrane. Here, we use a genetics approach to investigate and characterize determinants of spanin function. Because Rz1 is entirely embedded in the +1 reading frame of Rz, the genes were disembedded before using random mutagenesis to construct a library of lysis-defective alleles for both genes. Surprisingly, most of the lysis-defective missense mutants exhibited normal accumulation or localization in vivo, and also were found to be normal for complex formation in vitro. Analysis of the distribution and nature of single missense mutations revealed subdomains that resemble key motifs in established membrane-fusion systems, i.e., two coiled-coil domains in Rz, a proline-rich region of Rz1, and flexible linkers in both proteins. When coding sequences are aligned respective to the embedded genetic architecture of Rz1 within Rz, genetically silent domains of Rz1 correspond to mutationally sensitive domains in Rz, and vice versa, suggesting that the modular structure of the two subunits facilitated the evolutionary compression that resulted in the unique embedded gene architecture.

Phage Sepoy infects Salmonella enterica serovar Heidelberg, a Gram-negative bacterium that causes severe foodborne illnesses. Bacteriophages infecting this pathogen may be used as biocontrol agents for preventing Salmonella foodborne diseases. Here, we present the complete genome sequence of Sepoy, a T5-like siphophage.

Triple-negative breast cancer (TNBC) is the most invasive form of breast cancer due to an absence of estrogen (ER), progesterone (PR), and human epidermal growth factor-2 (HER2) receptors on the cell surface. TNBC accounts for approximately 12 to 20 percent of all breast cancer cases. The absence of ER, PR, and HER2 receptors on TNBCs and its ability to develop drug resistance renders it difficult to eradicate or retrogress tumor growth with hormonal therapy and chemotherapy. Triple-negative breast cancer is associated with poorer prognosis, increased chance of relapse, and lower chance of survival. Patients with TNBC have poorer outcome to conventional treatments than patients with other types of breast cancer. Natural killer cell-mediated immunotherapy is a promising therapeutic option for patients with TNBC. Natural killer cells contribute to the immune system by recognizing tumor cells through interactions between ligands on tumor cells and natural killer cell receptors. NK cell function is regulated by a net balance of signals from activating and inhibitory receptors interacting with ligands on target cells. Lectin-like Transcript-1 (LLT1, CLEC2D, OCIL) is a ligand that interacts with NK cell receptor NKRP1A (CD161) and inhibits NK cell activation. In this study, we have identified expression of LLT1 on TNBC cell lines MDA-MB-231 and MDA-MB-436 through flow cytometry, western blot, and confocal microscopy. We have demonstrated that blocking LLT1 on TNBCs with antibodies disrupts interaction with NKRP1A and enhances lysis of TNBCs by primary natural killer cells. We have also shown that a gene knockdown of LLT1 decreases cell surface expression of LLT1 on TNBCs and increases NK cell-mediated lysis of these TNBCs. The results suggest that LLT1 on TNBCs function as a method of evasion from immunosurveillance by NK cells. Blocking LLT1-NKRP1A interaction activates lysis by NK cells and will potentially open a new immunotherapeutic strategy for treatment of TNBC.