Bacteria
The paper aimed to study the mechanism by which gram-negative bacteria resist killing by antimicrobial peptides which are found in a diverse range of organisms and help restrict the growth of invading bacteria. Earlier studies have shown that gram-negative bacteria acquire resistance to antimicrobial peptides through modification in the lipid A portion of the cell surface. Lipid A modification is regulated by a two-component system called PhoPQ (Derzelle et al, 2004; Ernst et al, 1990; Guo et al, 1997; Moss et al, 200; Rebel et al, 2004). It is composed of a membrane-bound sensor kinase PhoQ and the cytosolic response regulator PhoP. The present study carried out several experiments to determine how bacterial sensor kinase recognizes antimicrobial peptides. Activation of the PhoPQ regulated gene was studied in varying Mg2+ concentrations. The experiment showed that while PhoP activity occurred at lower concentrations, it was impaired at higher concentrations suggesting that the antimicrobial peptide competes with Mg2+ which binds either with the bacterial cell surface or to PhoQ directly. Further experiment showed that a mutant strain in which phoQ was deleted failed to activate a PhoP-dependent gene suggesting that PhoQ is required for response to an antimicrobial peptide. Based on the experiments, the authors concluded that PhoQ is an antimicrobial peptide sensor and its acidic surface binds to divalent cations to repress kinase activity and to antimicrobial peptides to activate kinase activity. Based on the data obtained from the experiment, the paper proposes a mechanism for PhoQ activation. They suggest that the negatively charged surface of PhoQ is perfectly suited to sense the presence of antimicrobial peptide and binds with it. This functions as a lever to lift the acidic surface off the membrane which leads to signal transduction.
Strengths of the Paper
The paper fills an important gap in the understanding of how gram negative bacteria are able to resist the inherent immunity of most organisms. While previous studies have shown that the PhoPQ system responds to sub-lethal concentration of antimicrobial peptide, the exact mechanism was unknown. The paper builds on past researches which have shown antimicrobial peptides interact electrostatically with negatively charged outer membrane of lipid A (Piers and Hancock, 1994; Sawyer et al, 1988). The results of the experiments are also consistent with earlier works which have also proposed that Salmonella resistance to α-helical macrophage peptide is an important function of PhoPQ virulence promotion (Rosenberger et al, 2004).
Weaknesses and Suggestions for Improvement
The main problem with the paper is with way the data has been presented. The data obtained from the experiments has been shown graphically. A tabular representation of this data would have made it easier for the readers to understand and read it. Also the graphs are not properly labeled, making it very difficult to read them. For example figure 1 shows how antimicrobial peptides activate PhoPQ-dependent gene expression. But the graph does not have any legends making it very difficult to interpret it. Similarly, the results of the experiments are shown through the graphs in figure 5. These graph do not seem to be drawn to scale and give only a general idea of the results and not the accurate measurement. The explanation of figure 5b says that the fluorescence spectra wee recorded at the wavelength of 340nm, but the graph starts only at 400 nm. This discrepancy in the figure is not explained in the text either, suggesting that there is some mistake somewhere. Figure 5D is totally incomprehensible and superimposing the spectra of PhoQ in presence and absence of polymyxin nonapeptide does not serve any purpose. If the two spectra were presented in two separate graphs, it could have been easier to read them. Of course the best option would have been to tabulate the results to avoid confusion. One limitation of this paper is that it depends heavily on other papers and come to its conclusions based on indirect experiments rather than direct empirical studies.
Further Studies
Although there is a huge body of literature on the functioning of gram negative bacteria, the exact mechanism by which they are able to resist antimicrobial peptides is still not well understood. The present study attempts to find an answer based on experimental data. Although the results are consistent with past studies, the suggested mechanism is probably correct, future studies could try to directly observe the mechanism rather than come to conclusions based on indirect experiments. Also the paper does not study the mechanism by which PhoP promotes antimicrobial peptide resistance. Future studies could shed some more light on the role of PhoP in overcoming the antimicrobial peptides.
Trypanosomes
Akpan N., Caradonna, K., Chuenkova, M.V. and PereiraPerrin, M. (2008). Chagas’ disease parasite-derived neurotrophic factor activates cholinergic gene expression in neuronal PC12 cells. Brain Research. 1217. 195-202.
Summary of the Paper
The aim of the experiment was to study the affect of parasite-derived neurotropic factor (PDNF) produced by the parasite Trypanosoma cruzi, the parasite responsible for Chagas’ disease, on the expression of cholinergic gene in neuronal PC12 cells. In Chagas’ disease, PDNF binds with TrkA resulting in a multiple neurotrophic responses in neuronal cells. Given the importance of NGF/TrkA in triggering Chagas’ disease, the present study investigates if PDNF regulates the expression of choline acetyletransferase (ChAT) and vesicular ACh transporter (VAChT), proteins that define cholinergic phenotype in neurons. For the experiment, PC12 cells were treated with various concentrations of PDNF and analyzed for ChAt and VAChT. The results showed increased mRNA after 48 hours in both the proteins. Further experiments were conducted to investigate if T.cruzi invasion alters cholinergic gene expression by infecting PC12 cells with the parasite while all external parasites were removed. And later, the experiment was repeated with T.cruzi on the external surface while intracellular infection was removed. The experiment suggested that cholinergic gene expression is the result surface-mediated interactions between T.cruzi and host cell rather than intracellular infection. According to the authors’ PDNF stimulation of cholinergic gene expression could be responsible for recovery of parasympathetic function in Chagas’ disease. Based on these experiments, the authors conclude that PDNF could have therapeutic potential for Chagas’ disease.
Strengths of the Paper
The study added to the vast literature on the role of PDNF in activating cholinergic gene expression showing that Nerve Growth Factor (NGF) plays an important role in the maintenance of cholinergic phenotype (Lin et al, 2005; Tuszynski et al, 1990; Yeh et al, 1991; Auld et al, 2001; Ekstrom and Reinhold, 2004; Heisenberg et al, 1994). NGF’s biological activities is to a large part dependent on signaling through TrkA, a member of tyrosine receptor kinase superfamily (Counts and Mufson, 2005; Miller and Kaplan, 2001). The study also shows that the relative presence of T. cruzi infection with the cell determines whether this infection is beneficial or harmful for the organism. If the infection is present on the surface of the cell, it increases the expression of cholinergic gene while if the infection is intracellular, it decreases its expression. This finding could have important ramifications for future researches on cure for Chagas’ disease.
Weaknesses and Suggestions for Improvement
The authors suggest that PDNF stimulation of cholinergic gene could aid recovery in Chagas’ patients. However, experiments do not support this conclusion and authors’ explanation of how they arrived at this conclusion seems incomplete. The entire discussion section of the paper makes assumptions about the significance of the results of the experiment. There is almost no discussion of the main finding of the experiment which is that the presence of T.cruzi on the external cell wall increased cholinergic gene expression while intracellular infection actually decreased the gene expression. A detailed in-depth discussion of this finding and its ramifications could have been done in the discussion section. Also, the paper does not give any suggestions for future research.
Another problem with the paper is that the results are difficult to find and are interpolated with the method. If the results had been tabulated or placed separately from the methods, it would have been easy to understand their importance. In general, the layout of the paper is very confusing and it becomes very difficult to locate the various aspects of the experiment. Figure 4b shows the results of quantitative PCR, but the graph is not very clear. Also no mention is made of this graph anywhere in the text and seems to have been included as an afterthought.
Further Studies
Further studies could build on the findings of this paper and attempt to find cure for Chagas’ disease. Also, if the relative position of T.cruzzi cell can have completely opposite affect on gene expression, other parasites which influence gene expression could also show similar characteristics. This could be further studied and may help in finding cures for other diseases where a parasite affects gene expression.
Fungi
Legrand F., Lecuit, M., Dupont, B., Bellaton, E., Huerre, M., Rohrlich, P. and Lortholary, O. (2008) Adjuvant corticosteroid therapy for chronic disseminated candidiasis. Clinical Infectious Diseases. 46(5). 696-702.
Summary of the Paper
The aim of the experiment was to study the efficacy and tolerance of corticosteroid therapy (CST) in patients suffering from chronic disseminated candidiasis (CDC). Since CDC appears to share several features with immune reconstitution inflammatory syndrome, which has been effectively treated with CTC (French et al, 2004; Singh et al, 2005), it was hypothesized that the treatment would also be beneficial on CDC. The study reviewed the medical files of 6 adult and 4 minor leukemia patients who had CDC and were treated with CST. After a conformed diagnosis of CDC, patients were given age appropriate doses of CST in association with systemic antifungal therapy. The end point of the study was determined when all symptoms associated with CDC had been resolved. CST was administered for an average duration of 124.9 days and the findings of radiological analysis returned normal after a mean of 107 days. Clinical symptoms disappeared as early as 1 day. While the disappearance of clinical symptoms was resolved in record time in contrast to historical average exceeding 4 weeks (Thaler et al, 1988; Sallah et al, 1999 and Blade et al, 1992), the radiological abnormalities disappeared at around the same time as the historical average of 100 days (Kauffman et al, 1991). Based on these findings, the authors conclude that since clinical symptoms disappear so quickly, treatment for leukemia can be started earlier, thus giving timely treatment to the patients.
Strengths of the Paper
The study tries to find a way to cure CDC faster so that treatment for leukemia can be started without much delay. Not much research has been done in this area although several earlier studies have pointed out that the presence of CDC negatively impacts survival of leukemia patients (Anttila et al, 1997; Haron et al, 1987). Thus the paper tries to fill a gap in the existing research on this subject. The authors selected patients in five different hospitals and spread over a period of fifteen years, thus ensuring that the results were truly representative. All age groups, including children were included in the study. Also a wide spectrum of tests including biological, microbiological, radiological and liver biopsy was used to accurately diagnose CDC. The paper also notes in details patient characteristics before and after the onset of CDC, and the exact time gaps between the various occurrences such as onset of CDC and its cure. The authors also follow-up the patients over several years following treatment to ensure that there is no relapse and that the treatment has had desired impact. However, their conclusion that CDC must belong to IFI-induced IRIS seems to be without much support.
Weaknesses and Suggestions for Improvement
Although the study shows that CST can cure clinical symptoms of CDC as early as one day, the symptoms of the disease remain for an average of 100 days. The study does not discuss the implications of this and how it may adversely impact the patients. Also, the authors mention a higher median survival rate of 40.8% than historical rate of 26%. This implies that the increased survival rate is a direct result of CST treatment. However, the authors have neither provided the data nor tried to substantiate this claim in any way. The higher survival rate could be due to any number of reasons which have not been studied. The earlier study had taken place at a time when many of technologies now available were not available. The paper should have carried out a comparison of the treatment given in the historical study to the present treatment before making the claim.
Also, in the discussion, it is mentioned that since CDC responded to CST treatment it must belong to IFI-induced IRIS. This statement has not been properly explained and seems like the authors are jumping to an unwarranted conclusion.
Further Studies
Overall, the study comes up with a treatment for CDC which would help patients get treatment for leukemia in time and thus prevent unnecessary deaths. This will definitely benefit many patients. However, Further study needs to be carried out on the nature of CDC to understand the cause of this disease so that it can be completely eradicated in a shorter time than the present average of 100 days.
Apicomplexa
Kafsack BF, Pena JD, Coppens I, Ravindran S, Boothroyd JC and Carruthers, VB. (2009). Rapid membrane disruption by a perforin-like protein facilitates parasite exit from host cells. Science. 323(5913): 530-533.
Summary of the Paper
The aim of the paper is to study the role of Toxoplasma perforin-like protein 1 (TgPLP1) and understand the mechanism which helps the parasite Toxoplasma gondii egress the host cell at the end of its growth period. Toxoplasma is among the organisms that have perforin like protein (PLP). Proteins in this domain, known as the Membrane Attack Complex Perforin (MACPF), induce cell death by oligomerizing on the surface of target cells and inserting to form large pores (Pipkin and Lieberman, 2007). The MACPF domain of TgPLP1 is very similar to MACPF domain of mammals, bacteria and protozoa and exhibits core sequence which is very important for pore formation. The toxoplasma genome contains two MACPF gene (plp1 and plp2). However, plp2 does not express itself. The authors deleted the plp1 gene thus preventing the expression of TgPLP1 in plp1ko. While in malaria, similar genetic modification failed to have any impact, in Toxoplasma, absence of TgPLP1 meant that at the end of its 48 hours growth period, the parasite was unable to egress from the host cell. When mice were infected with plp1ko an wild type (WT) pathogens, those infected with just 10 WT tachyzoites died within 15 days while those infected with up to 1 million plp1ko tachyzoites survived. Based on the study, the authors conclude plp1 is responsible for parasite egression and that unlike in malaria, despite the presence of TgPLP1, the mechanical disruption of the parasitophorous vacuolar membrane (PVM) and host cell plasma membrane (HPM) is due to gliding motility and not due to pore formation.
Strengths of the Paper
The paper brings out an important difference between malaria and toxoplasma parasites, a parasite which causes congenital birth defect, ocular disease and encephalitis in immunocompromised individuals (Montoya and Liesenfeld, 2004). The genetically modified Toxoplasma are compared to the wild type and to Toxoplasma with slightly below normal expression of TgPLP1 (plp1ko/PLP1myc). These comparisons made it clear that the plp1 gene is responsible for helping the parasite egress the host cell. The experiments on mice proved that plp1 gene is affects the virulence of the parasite. Also, unlike in malaria, it is not the pore formation but the gliding motility which aids the egress and kills the host cell. However, the paper remains silent on what causes death of the organism.
Weaknesses and Suggestions for Improvement
While the experiment showed that TgPLP1 compromised the integrity of the membrane of the host cell, it was unable to explain the mechanism by which this happens. The study shows that the absence of plp1 gene makes it difficult for the organism to egress the host cell. It also shows that gliding motility instead of pore formation helps the parasite egress. But it does not explain how the integrity of the host cell is compromised and how gliding motility compromises the cell membrane. When the plp1 gene was absent, the parasites still tried to egress by prodding and deforming the membrane, they were not successful but the WT tachyzoites faced no resistance. The paper fails to explain, or tries to understand the chemistry behind this failure to egress. We do not know the chemical or biological changes that take place which prevent the parasite from egression in the absence of plp1.
The paper also does not discuss the implications of their findings. There is no mention of how future researches may be able to benefit from this experiment or any advice for future research. The final conclusion stating that a protein analogous to TgPLP1 could be responsible helping malaria parasite egress from infected erythrocytes is entirely unsubstantiated and should have been discussed in more details.
Finally, authors make use of many pictures and graphical representations to explain their experiment but some of these pictures are not very clear. For example, figure 3A tries to show pore formation but nothing is visible in the pictures, despite the authors having marked them clearly. The authors could have supplemented these pictures with diagrams to give a better understanding of the procedures.
Further Studies
The paper identifies the role of TgPLP1 in Toxoplasma, though it remains silent on how this fact may be exploited. Future studies could investigate the implications o the findings of this paper and could even try and search for ways to reduce the virulence of toxoplasma so that infected individuals could be treated. Also, the role of PLP in malaria parasite could also be studied in details to substantiate the authors’ suggestion that PLP in malaria would have a function analogous to TgPLP1.
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