Increased Incidence of Colon Tumors in AOM-Treated Apc 1638N/+ Mice Reveals Higher Frequency of Tumor Associated Neutrophils in Colon Than Small Intestine.

Colorectal cancer (CRC) is one of the most common cancers and a major cause of mortality. Mice with truncating Apc germline mutations have been used as a standard model of CRC, but most of the Apc-mutated lines develop multiple tumors in the proximal small intestine and rarely in the colon precluding detailed analysis of colon tumor microenvironment. Our aim was to develop a model with higher resemblance to human CRC and to characterize tumor infiltrating immune cells in spontaneously developing colon tumors compared to small intestinal tumors. Therefore, the Apc 1638N/+ line was treated repeatedly with azoxymethane (AOM) and 90% colon tumor incidence and 4 to 5 colon tumors per mouse were achieved. Of note, AOM treatment specifically increased the tumor burden in the colon, but not in the small intestine. Histological grading and WNT-signaling activity did not differ significantly between small intestinal and colon tumors with some lesions progressing to invasive adenocarcinoma in both locations. However, characterization of the intratumoral myeloid cell compartment revealed a massive infiltration of colon tumors with neutrophils - 6-fold higher than in small intestinal tumors. Moreover, CCL17-expressing macrophages and dendritic cells accumulated in the tumors indicating the establishment of a tumor-promoting immunosuppressive environment. Thus, Apc 1638N/+ mice treated with AOM are a suitable and straightforward model to study the influence of immune cells and chemokines on colon carcinogenesis.

CsrA modulates levels of lipoproteins and key regulators of gene expression critical for pathogenic mechanisms of Borrelia burgdorferi.

Carbon storage regulator A (CsrA) is an RNA binding protein that has been characterized in many bacterial species to play a central regulatory role by modulating several metabolic processes. We recently showed that a homolog of CsrA in Borrelia burgdorferi (CsrA(Bb), BB0184) was upregulated in response to propagation of B. burgdorferi under mammalian host-specific conditions. In order to further delineate the role of CsrA(Bb), we generated a deletion mutant designated ES10 in a linear plasmid 25-negative isolate of B. burgdorferi strain B31 (ML23). The deletion mutant was screened by PCR and Southern blot hybridization, and a lack of synthesis of CsrA(Bb) in ES10 was confirmed by immunoblot analysis. Analysis of ES10 propagated at pH 6.8/37°C revealed a significant reduction in the levels of OspC, DbpA, BBK32, and BBA64 compared to those for the parental wild-type strain propagated under these conditions, while there were no significant changes in the levels of either OspA or P66. Moreover, the levels of two regulatory proteins, RpoS and BosR, were also found to be lower in ES10 than in the control strain. Quantitative real-time reverse transcription-PCR analysis of total RNA extracted from the parental strain and csrA(Bb) mutant revealed significant differences in gene expression consistent with the changes at the protein level. Neither the csrA(Bb) mutant nor the trans-complemented strain was capable of infection following intradermal needle inoculation in C3H/HeN mice at either 10³ or 105 spirochetes per mouse. The further characterization of molecular basis of regulation mediated by CsrA(Bb) will provide significant insights into the pathophysiology of B. burgdorferi.

Overexpression of CsrA (BB0184) alters the morphology and antigen profiles of Borrelia burgdorferi.

Borrelia burgdorferi, the agent of Lyme disease, alters its gene expression in response to highly disparate environmental signals encountered in its hosts. Among the relatively few regulators of adaptive gene expression present in the borrelial genome is an open reading frame (ORF), BB0184, annotated as CsrA (carbon storage regulator A). CsrA, in several bacterial species, has been characterized as a small RNA binding protein that functions as a global regulator affecting mRNA stability or levels of translation of multiple ORFs. Consistent with known functions of CsrA, overexpression of CsrA from B. burgdorferi (CsrABb) in Escherichia coli resulted in reduced accumulation of glycogen. We determined that csrABb is part of the flgK motility operon and that the synthesis of CsrABb was increased when B. burgdorferi was propagated under fed-tick conditions. Overexpression of CsrABb in B. burgdorferi strain B31 (ML23, lp25-negative clonal isolate) resulted in a clone, designated ES25, which exhibited alterations in colony morphology and a significant reduction in the levels of FlaB. Several lipoproteins previously characterized as playing a role in infectivity were also altered in ES25. Real-time reverse transcription-PCR analysis of RNA revealed significant differences in the transcriptional levels of ospC in ES25, while there were no such differences in the levels of other transcripts, suggesting posttranscriptional regulation of expression of these latter genes. These observations indicate that CsrABb plays a role in the regulation of expression of pathophysiological determinants of B. burgdorferi, and further characterization of CsrABb will help in better understanding of the regulators of gene expression in B. burgdorferi.

Deletion of BBA64, BBA65, and BBA66 loci does not alter the infectivity of Borrelia burgdorferi in the murine model of Lyme disease.

Borrelia burgdorferi, the causative agent of Lyme disease, alters its gene expression in response to highly disparate environmental signals encountered in its tick vector versus vertebrate hosts. Whole-genome transcriptional profile analysis of B. burgdorferi, propagated in vitro under mammalian-host-specific conditions, revealed significant upregulation of several linear plasmid 54 (lp54)-encoded open reading frames (ORFs). Among these ORFs, BBA64, BBA65, and BBA66 have been shown to be upregulated in response to multiple mammalian-host-specific signals. Recently, we determined that there was no significant difference in the ability of BBA64(-) mutant to infect C3H/HeN mice compared to its isogenic control strains, suggesting that B. burgdorferi might utilize multiple, functionally related determinants to establish infection. We further generated BBA65(-) and BBA66(-) single mutants in a noninfectious, lp25(-) clonal isolate of B. burgdorferi strain B31 (ML23) and complemented them with the minimal region of lp25 (BBE22) required for restoring the infectivity. In addition, we generated a BBA64(-) BBA65(-) BBA66(-) triple mutant using an infectious, clonal isolate of B. burgdorferi strain B31 (5A11) that has all of the infection-associated plasmids. There were no significant differences in the ability to isolate viable spirochetes from different tissues of C3H/HeN mice infected via intradermal needle inoculation with either the individual single mutants or the triple mutant compared to their respective isogenic parental strains at days 21 and 62 postinfection. These observations suggest that B. burgdorferi can establish infection in the absence of expression of BBA64, BBA65, and BBA66 in the murine model of Lyme disease.

Role of the BBA64 locus of Borrelia burgdorferi in early stages of infectivity in a murine model of Lyme disease.

Borrelia burgdorferi, the causative agent of Lyme disease, undergoes rapid adaptive gene expression in response to environmental signals encountered during different stages of its life cycle in the arthropod vector or the mammalian host. Among all the plasmid-encoded genes of B. burgdorferi, several linear plasmid 54 (lp54)-encoded open reading frames (ORFs) exhibit the greatest differential expression in response to mammalian host-specific temperature, pH, and other uncharacterized signals. These ORFs include members of the paralogous gene family 54 (pgf 54), such as BBA64, BBA65, and BBA66, present on lp54. In an attempt to correlate transcriptional up-regulation of these pgf 54 members to their role in infectivity, we inactivated BBA64 and characterized the phenotype of this mutant both in vitro and in vivo. There were no major differences in the protein profiles between the BBA64 mutant and the control strains, while immunoblot analysis indicated that inactivation of BBA64 resulted in increased levels of BBA65. Moreover, there was no significant difference in the ability of the BBA64 mutant to infect C3H/HeN mice compared to that of its parental or complemented control strains as determined by culturing of viable spirochetes from infected tissues. However, enumeration of spirochetes using quantitative real-time PCR revealed tissue-specific differences, suggesting a minimal role for BBA64 in the survival of B. burgdorferi in select tissues. Infectivity analysis of the BBA64 mutant suggests that B. burgdorferi may utilize multiple determinants to establish infection in mammalian hosts.