Nankai Feng Lu/Wang Lei's team discovered a new mechanism to effectively inhibit pathogenic Escherichia coli to cause urinary tract infection Cell Press Dialogue with Scientists

Life science On April 19, 2022, the team of Feng Lu and Wang Lei of Nankai University published a research titled "Bladder Epithelial Cell Phosphate Transporter Inhibition Protects Mice Against Uropathogenic Escherichia coli Infection" in Cell Reports, a journal of Cell Press.
In this paper, the study revealed the molecular mechanism of the key vesicle escape link in the pathogenic process of uropathogenic Escherichia coli (UPEC), and found that the use of Accell siRNA technology to interfere with the host phosphate transporter PIT1 can effectively control UPEC caused urinary tract infection
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▲Long press the picture to identify the QR code to read the original text Urinary tract infection (UTI) causes serious disease, with a large number of infections (second only to respiratory and digestive tract infections) and repeated infections (even after antibiotic treatment, there are still 30-50% of patients have recurrent seizures), causing lifelong pain to many patients and a great burden to medical resources
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75-95% of UTIs are caused by UPEC
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UPEC first infects bladder epithelial cells through fusiform vesicles unique to bladder epithelial cells
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Fusiform vesicles are characterized by exocytosis in both physiological and immune functions
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To avoid being cleared by exocytosis, UPECs need to escape from the fusiform vesicles into the cytoplasm and replicate massively on a monoclonal basis to form intracellular bacterial communities (IBCs) containing more than 10,000 bacteria
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The formation of IBC allows UPEC to successfully colonize bladder epithelial cells and cause disease, and also enhances the drug resistance of the bacteria
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Therefore, the escape of UPEC from fusiform vesicles into the cytoplasm is a key link in its pathogenic process, but the molecular mechanism is currently unclear
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Using cell models, mouse models, and molecular biology, the team of Feng Lu and Wang Lei discovered that UPECs sense the host immune response and express an outer membrane phospholipase PldA to destroy the host's fusiform vesicle membrane and mediate its escape
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UPEC infection upregulates the expression of the host cell's phosphate transporter PIT1 through the TLR4/NF-κB pathway
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PIT1 is located on the fusiform vesicle membrane and transports phosphate into the cytoplasm
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Upregulation of PIT1 expression reduces phosphate concentration within fusiform vesicles
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PldA of UPEC mediates the escape of UPEC from the fusiform vesicle into the cytoplasm by disrupting the vesicle membrane in response to the low-concentration inorganic phosphorus environment in the fusiform vesicle through the two-component system PhoBR
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Knockdown of PIT1 in vivo blocked the escape of UPEC from fusiform vesicles in bladder epithelial cells, inhibited the formation of IBCs in mouse bladder, and protected mice from UPEC infection
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Urinary tract infection is a multiple common disease that seriously affects the quality of life of patients.
The new findings of this study on its pathogenic mechanism provide new ideas for the treatment of this chronic disease.
Cell Reports was also published from Baylor Medicine, USA.
Professor Indira Mysorekar's review article entitled "PITing it forward: A new link in the journey of Uropathogenic E.
coli in the urothelium" pointed out the important theoretical innovation and application value of this discovery, and proposed that the future can be achieved through Develop small drug molecules targeting this pathogen-host interaction mechanism to treat and prevent UTI
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The author's interview with the official account of Cell Press specially invited Professor Feng Lu to accept the interview on behalf of the research team, and asked her to interpret it in detail
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CellPress: Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs)
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What are the current treatment options for UTIs? What problems remain? Prof.
Feng Lu: The current treatment options for acute urinary tract infections are mainly antibiotics, and clinically, trimethoprim-sulfamethoxazole and quinolones antibiotics are mainly used to treat UTIs
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However, antibiotic treatment does not completely clear UPECs in the bladder and therefore does not suppress recurrent infections
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At the same time, the existence of a large number of UPEC-resistant strains also limits the application of antibiotics
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The massive replication of UPEC in the cytoplasm to form an intracellular bacterial community (IBC) containing more than 10,000 bacteria is the main reason why antibiotics cannot completely remove UPEC
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IBCs are biofilm-like bacterial communities that are difficult for antibiotics to fully penetrate
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In addition, IBC formation is a multi-cycle process, that is, from a single clone to the release of a large number of bacteria from bladder epithelial cells after IBC maturation to complete a cycle
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The released bacteria infect new bladder epithelial cells and begin the next cycle, during which some bacteria can invade the underlying bladder epithelial cells and lie dormant in vesicles, a state known as a quiescent intracellular reservoir (QIR).

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Once host immunity declines, bacteria in QIR can cause a new round of infection
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The formation and repeated infection of IBCs in the acute phase lead to high drug resistance of UPECs, which brings difficulties to the complete cure of UTIs
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CellPress: Vesicle escape of UPECs is a key mechanism for their escape from host cell exocytosis
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Which factors play a key role in the escape of UPEC vesicles? Prof.
Feng Lu: UPEC first needs to sense the signals in the host vesicle, and secondly, it needs to activate the corresponding escape pathway
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Active exocytosis of invading bacteria to the outside of cells is one of the innate immune defense strategies of bladder epithelial cells
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We found that activation of innate immunity in bladder epithelial cells by UPEC leads to a decrease in the level of phosphorus in vesicles, and UPEC can sense the low phosphorus environment in vesicles through the two-component system PhoBR and use this signal to induce the expression of an outer membrane phospholipid The enzyme PldA degrades vesicle membrane phospholipids to help them escape from the vesicle into the cytoplasm, thereby evading host cell exocytosis
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CellPress: What is the expression pattern of the phosphate transporter PIT1 in the fusiform vesicles of bladder epithelial cells (BECs)? What is the role of PIT1 in the expression of UPEC PldA? Prof.
Feng Lu: PIT1 is a phosphorus transporter located in the outer membrane of host cells, responsible for transporting extracellular inorganic phosphorus molecules into the cytoplasm
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Since fusiform vesicles are formed by the invagination of the adventitial membrane of bladder epithelial cells (BECs), PIT1 located in the outer membrane of the vesicles transports phosphorus molecules from the vesicles outward
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PldA is a phospholipase located in the outer membrane of bacteria and has the function of cleaving eukaryotic cell membranes
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We found that when UPECs infect BECs via fusiform vesicles, the expression of PIT1 is up-regulated through the TLR4/NF-κB immune activation pathway, which transports vesicle inorganic phosphate molecules into the cytoplasm, resulting in the concentration of inorganic phosphate in fusiform vesicles decrease
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In order to avoid being cleared by exocytosis, UPEC in the vesicle induces the expression of PldA by sensing the decrease in the concentration of inorganic phosphate in the fusiform vesicle and using this as a signal to help it escape from the fusiform vesicle to the cytoplasm to replicate in large quantities IBCs are formed, causing disease
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Therefore, the expression of PIT1 as the origin of the low phosphorus signaling molecule in the vesicle plays a crucial role in inducing the expression of PldA in UPEC
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CellPress: How does disrupting the expression of PIT1 affect UPEC infection? Prof.
Feng Lu: Our study shows that interfering with host PIT1 expression can prevent UPECs from escaping from fusiform vesicles into the cytoplasm, allowing them to be more exocytosed out of cells by host cells, thereby reducing the formation of IBCs in mouse bladder cells.
Reduce the colonization and dissemination of UPEC in mouse bladder tissue, thereby inhibiting the occurrence of urinary tract infection
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We also found that interfering with PIT1 expression in a timely manner after the onset of infection could inhibit the recurrence of infection
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CellPress: This study reveals the mechanism by which UPEC escapes host cell exocytosis.
What guidance can this provide for the treatment of UPEC infection? Prof.
Feng Lu: After UPEC infects bladder epithelial cells through fusiform vesicles, in order to escape the exocytosis of host cells, it needs to escape from the vesicles to the cytoplasm for replication and colonization
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Thus preventing UPEC from escaping from the vesicle and subsequent infection does not occur
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Our study revealed the molecular mechanism of UPEC escape from fusiform vesicles, a key step in bacterial pathogenesis mediated by host protein PIT1, and demonstrated in mice using Accell siRNA technology that disrupting PIT1 expression could reduce UPEC in mouse bladder tissue colonization and inhibit the occurrence of urinary tract infections
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Accell siRNA is a mature RNA interference therapy that can be applied to humans
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Therefore, our work not only discovered a new drug target, but also proved the feasibility of using more mature technology to achieve treatment
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CellPress: This study found that UPEC can use the host's immune response to assist its own vesicle escape, which provides a new understanding for us to understand the interaction mode between pathogenic bacteria and the host.
What aspects and methods will be considered in the future? Further in-depth study of its entire biological process? Prof.
Feng Lu: The relationship between pathogenic bacteria and the host immune system is like an arms race
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The immune systems of pathogenic bacteria and host cells have co-evolved over millions of years.
For pathogenic bacteria, they must be able to escape the host's immune surveillance at all stages of host infection to successfully cause disease
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We found that UPECs use the host's innate immune response to assist themselves in escaping vesicles and avoid exocytosis by host cells, which is one of the means by which UPECs escape host cell immune surveillance
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UPECs that escape into the cytoplasm still have to escape the immune surveillance in the host cytoplasm (such as inflammasome-mediated pyroptosis) and then proliferate, and the relevant molecular mechanisms are currently poorly understood
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In the future, we will further use cell biology, immunology, molecular biology and other methods to explore how UPEC evades immune surveillance in the host cytoplasm to help its pathogenesis
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About the Author Prof.
Feng Lu The corresponding author of the paper, Prof.
Feng Lu received a bachelor's degree and a doctorate degree from Jiangnan University and the University of Sydney, respectively
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He is currently a professor at TEDA Institute of Biotechnology, Nankai University
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In 2008, he was awarded the National Fund for Distinguished Young Scholars, and in 2009, he was selected as the Yangtze River Scholar of the Ministry of Education
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Main research directions: 1) The pathogenic mechanism of intestinal pathogens; 2) The interaction mechanism between pathogens and the host
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He has published 171 SCI papers in PNAS, Cell Reports, Nature Communications and other journals, with a total impact factor of 555
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Prof.
Lei Wang Corresponding author of the paper, Prof.
Lei Wang is currently the vice president of Nankai University
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Nankai University and Sydney University received bachelor's and doctoral degrees, respectively
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In 2001, he was selected as a Changjiang Scholars Distinguished Professor by the Ministry of Education, won the National Science Fund for Distinguished Young Scholars in 2002, and was selected into the "Ten Thousand People Program" of the Organization Department of the Central Committee in 2014
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He is mainly engaged in the research on the molecular evolution and pathogenic mechanism of pathogenic bacteria, and the interaction between pathogenic bacteria and the host.
He has published 255 SCI papers in journals such as Nature, PNAS, and Nature Communications, with an H factor of 50
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Pang Yu The first author of the doctoral dissertation, Pang Yu, graduated from Tianjin University of Traditional Chinese Medicine with a bachelor's degree in clinical pharmacy, and a Ph.
D.
degree in microbiology from Nankai University, under the tutelage of Professor Feng Lu
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Now Nankai University is conducting postdoctoral research, the main research direction is the research on the pathogenesis of uropathogenic Escherichia coli, and has been funded by the Postdoctoral Science Fund
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The first author of Cheng Zhihui's doctoral dissertation, Cheng Zhihui, obtained his undergraduate degree and master of science degree from Nankai University in 1999 and 2002, and then entered the Ohio State University, where he studied with Prof.
Yasuko Rikihisa, an academician of the National Academy of Sciences, and obtained his doctorate degree in 2008.
He graduated Continue to do postdoctoral research at Ohio State University
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Currently employed in the Department of Microbiology of Nankai University, his main research directions are the pathogenic mechanism of pathogenic bacteria, the research and development of new antibacterial drugs, and the immune response of the host.
He has received many grants from the National Natural Science Foundation of China
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Zhang Si, the first author of the doctoral dissertation, Zhang Si, Ph.
D.
student of TEDA Institute of Biotechnology, Nankai University, graduated from Hubei Medical College with a bachelor’s degree in clinical medicine, and a master’s degree in pathogen biology from Lanzhou University, and entered TEDA Biotechnology, Nankai University in 2017 The Institute, under the tutelage of Professor Wang Lei, is dedicated to the research on the pathogenic mechanism of uropathogenic Escherichia coli
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He was awarded the "Yun Gong Yun Neng Second-Class Scholarship" of Nankai University
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Related paper information The original paper was published in Cell Reports, a journal of CellPress Cell Press.