Design and synthesis of broad-spectrum antimicrobial amphiphilic peptidomimetics to combat drug-resistance.

The gradual depletion of antibiotic discovery pipeline makes the antibiotic resistance a difficult clinical problem and a global health emergency. The membrane-active antimicrobial peptides (AMPs) attracted much attention due to a lower tendency to bacterial resistance than traditional antibiotics. However, some immanent drawbacks of AMPs may hamper their application in combating antibiotic resistance in the long run, such as susceptible to enzymatic degradation and low cell permeability. Herein, we report the design and synthesis of a novel series of amphiphilic peptidomimetics, from which we identified compounds that exhibited potent antimicrobial activity against a panel of clinically relevant Gram-positive and Gram-negative bacteria strains. The most potent compound 20 (SD-110-12) is able to kill intracellular bacterial pathogens and prevent the development of bacterial resistance under the tested conditions by targeting cell membranes. Additionally, compound 20 (SD-110-12) obtains good in vivo efficacy that is comparative to vancomycin by eradicating MRSA and suppressing inflammation in a mice infected skin wound model, demonstrating its promising therapeutic potential.

Characteristics of inflammatory reactions during development of liver abscess in hamsters inoculated with Entamoeba nuttalli.

BACKGROUND: Entamoeba nuttalli is an intestinal protozoan with pathogenic potential that can cause amebic liver abscess. It is highly prevalent in wild and captive macaques. Recently, cysts were detected in a caretaker of nonhuman primates in a zoo, indicating that E. nuttalli may be a zoonotic pathogen. Therefore, it is important to evaluate the pathogenicity of E. nuttalli in detail and in comparison with that of E. histolytica. METHODOLOGY/PRINCIPAL FINDINGS: Trophozoites of E. nuttalli GY4 and E. histolytica SAW755 strains were inoculated into liver of hamsters. Expression levels of proinflammatory factors of hamsters and virulence factors from E. histolytica and E. nuttalli were compared between the two parasites. Inoculations with trophozoites of E. nuttalli resulted in an average necrotic area of 24% in liver tissue in 7 days, whereas this area produced by E. histolytica was nearly 50%. Along with the mild liver tissue damage induced by E. nuttalli, expression levels of proinflammatory factors (TNF-α, IL-6 and IL-1ß) and amebic virulence protein genes (lectins, cysteine proteases and amoeba pores) in local tissues were lower with E. nuttalli in comparison with E. histolytica. In addition, M2 type macrophages were increased in E. nuttalli-induced amebic liver abscesses in the late stage of disease progression and lysate of E. nuttalli trophozoites induced higher arginase expression than E. histolytica in vitro. CONCLUSIONS/SIGNIFICANCE: The results show that differential secretion of amebic virulence proteins during E. nuttalli infection triggered lower levels of secretion of various cytokines and had an impact on polarization of macrophages towards a M1/M2 balance. However, regardless of the degree of macrophage polarization, there is unambiguous evidence of an intense acute inflammatory reaction in liver of hamsters after infection by both Entamoeba species.

Evaluation of the C-Terminal Fragment of Entamoeba histolytica Gal/GalNAc Lectin Intermediate Subunit as a Vaccine Candidate against Amebic Liver Abscess.

BACKGROUND: Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis, including amebic dysentery and liver abscesses. E. histolytica invades host tissues by adhering onto cells and phagocytosing them depending on the adaptation and expression of pathogenic factors, including Gal/GalNAc lectin. We have previously reported that E. histolytica possesses multiple CXXC sequence motifs, with the intermediate subunit of Gal/GalNAc lectin (i.e., Igl) as a key factor affecting the amoeba's pathogenicity. The present work showed the effect of immunization with recombinant Igl on amebic liver abscess formation and the corresponding immunological properties. METHODOLOGY/PRINCIPAL FINDINGS: A prokaryotic expression system was used to prepare the full-length Igl and the N-terminal, middle, and C-terminal fragments (C-Igl) of Igl. Vaccine efficacy was assessed by challenging hamsters with an intrahepatic injection of E. histolytica trophozoites. Hamsters intramuscularly immunized with full-length Igl and C-Igl were found to be 92% and 96% immune to liver abscess formation, respectively. Immune-response evaluation revealed that C-Igl can generate significant humoral immune responses, with high levels of antibodies in sera from immunized hamsters inhibiting 80% of trophozoites adherence to mammalian cells and inducing 80% more complement-mediated lysis of trophozoites compared with the control. C-Igl was further assessed for its cellular response by cytokine-gene qPCR analysis. The productions of IL-4 (8.4-fold) and IL-10 (2-fold) in the spleen cells of immunized hamsters were enhanced after in vitro stimulation. IL-4 expression was also supported by increased programmed cell death 1 ligand 1 gene. CONCLUSIONS/SIGNIFICANCE: Immunobiochemical characterization strongly suggests the potential of recombinant Igl, especially the C-terminal fragment, as a vaccine candidate against amoebiasis. Moreover, protection through Th2-cell participation enabled effective humoral immunity against amebic liver abscesses.

Comparative analysis of genotypic diversity in Entamoeba nuttalli isolates from Tibetan macaques and rhesus macaques in China.

We have recently demonstrated the potentially virulent species Entamoeba nuttalli as one of the highly prevalent parasites in rhesus macaques (Macaca mulatta) in Mount Long-hu and Gui-yang in China. Tibetan macaque (Macaca thibetana) is a unique species living in China. To evaluate the prevalence of Entamoeba species in wild Tibetan macaques, we obtained 89 stool samples in Mount E-mei of Si-chuan Province in China. PCR analysis detected E. nuttalli, Entamoeba coli, and Entamoeba polecki ST2 in 17%, 42%, and 66% of the samples, respectively, whereas Entamoeba histolytica and Entamoeba dispar were undetected. This study is the first to report on the detection of E. nuttalli from Tibetan macaques. Six E. nuttalli isolates were obtained, 18S rRNA gene and six tRNA-linked short tandem repeat (STR) loci of the isolates were sequenced. The Mantel test results gave an r value of 0.97 of relationships between geographical distance and genetic diversity of Chinese E. nuttalli populations, indicating a significant isolation-by-distance effect in Chinese E. nuttalli according to the tRNA-STR loci sequences. Structural analysis of E. nuttalli isolates based on tRNA-linked STR loci demonstrated three Chinese E. nuttalli populations with their respective features, but the Gui-yang population was located in the middle. In the distance-based NJ tree, E. nuttalli isolates were divided into five different branches, and E-mei isolates were attributed to an independent branch to distinguish them from Gui-yang and Long-hu isolates. Genetic analysis in this study provided clues of the genetic differences between E. nuttalli isolates from Tibetan macaques and rhesus macaques in China.

Amebic lung abscess with coexisting lung adenocarcinoma: a unusual case of amebiasis.

Amebic lung abscess with concurrent lung cancer, but without either a liver abscess or amebic colitis, is extremely uncommon. Here, we report a 70-year-old man presenting with pulmonary amebiasis and coexisting lung adenocarcinoma. During his first-time hospitalization, the diagnosis of lung amebiasis was confirmed by morphological observation and PCR in formalin-fixed and paraffin-embedded sediments of pleural effusion. Almost four months later, the patient was readmitted to hospital for similar complaints. On readmission, lung adenocarcinoma was diagnosed by liquid-based sputum cytology and thought to be delayed because coexisting amebic lung abscess. This case demonstrated that sediments of pleural effusion may be used for further pathological examination after routine cytology has shown negative results. At the same time, we concluded that lung cancer may easily go undetected in the patients with pulmonary amebiasis and repetitive evaluation by cytology and imaging follow-up are useful to find potential cancer.

Primary pathogenicity analysis of a Chinese Entamoeba histolytica isolate.

This study is the first to isolate an Entamoeba histolytica strain from Chinese amoebic patients and to conduct a detailed examination of its virulence. A fecal sample that contains cysts of E. histolytica was obtained from Guangxi province. The sample was cultured axenically and then cloned by limiting dilution, and named as XLAC. In vitro and in vivo tests were conducted to evaluate the virulence of the Entamoeba isolate. The E. histolytica strain XLAC was successfully cloned and cultured axenically. DNA regions that contain hexokinase, glucose-6-phosphate isomerase, phosphoglucomutase, and heavy subunit of lectin genes were amplified by PCR. The PCR products were then sequenced. Virulence analysis suggested that the XLAC strain was similar to the HM1:IMSS strain at the genetic level. In vitro and in vivo tests also implicated these strains to be similar. These findings may be attributed to the low expression levels of pathogenic genes obtained through realtime PCR. The XLAC strain restored its virulence after it was injected into hamster liver. This study may be a good model for studying virulence changes in E. histolytica.

Prevalence and genetic diversity of Entamoeba species infecting macaques in southwest China.

Many colonies of macaques (Macaca fascicularis and Macaca mulatta) are maintained in China, especially in Guangxi and Guizhou. A total of 803 fresh stool samples infected with Entamoeba were obtained from three big colonies of macaques located in southwest China. The samples were examined for the presence of five Entamoeba species using PCR. Entamoeba nuttalli, Entamoeba dispar, Entamoeba coli, and Entamoeba chattoni infections were detected, but Entamoeba histolytica infection was not. This study is the first to report on the prevalence of E. nuttalli in wild macaques from China. Eighteen E. nuttalli isolates and five E. dispar isolates were obtained by culturing the samples in Tanabe-Chiba medium. The serine-rich protein (SRP), ribosomal RNA (rRNA), hexokinase (HXK), glucose-6-phosphate isomerase (GPI), and phosphoglucomutase (PGM) genes of E. nuttalli isolates were compared with other reported isolates. The results showed clear differences among the Chinese E. nuttalli isolates and other isolates based on the SRP gene sequences. However, HXK, GPI, and PGM genes of these strains were similar to those of other isolates. The rRNA genes of E. coli and E. chattoni were also amplified and analyzed from these samples. The results suggested that host species might be a more important factor than geographic location in amebic genetic diversity.

Unique short tandem repeat nucleotide sequences in Entamoeba histolytica isolates from China.

A few PCR-based DNA typing methods using repetitive elements contained within both protein-coding genes and noncoding DNAs have been reported for Entamoeba histolytica over the years. The serine-rich E. histolytica protein and tRNA-linked short tandem repeats (STRs) are most commonly used to investigate the relationship between parasite genotype and E. histolytica infection outcome. Many E. histolytica infections in China have been reported; however, little genome information has been provided. In the current paper, five Chinese E. histolytica samples were reported: three amoebic liver abscess cases, one combined case and one asymptomatic case. Our study is the first to report on the DNA typing information of E. histolytica in China. We included two city, one imported, and two country cases. Sequence analysis of serine-rich protein genes confirmed the presence of seven sequence types in five isolates. The STRs amplified from the samples revealed five STR variations in the A-L, four in the N-K2, and R-R loci, three in D-A, S(TGA)-D and S-Q loci. Two country patients were found to have a different outcome of infection with the same genotypes of E. histolytica, whereas in a city case, one E. histolytica strain had led to different outcome of the infection in one patient. Analyses of the results suggest that more genome information of E. histolytica strains from China through accurate methods is needed to interpret how the parasite genome plays a role in determining the outcome of E. histolytica infections.